Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.     

Avicins, natural anticancer saponins, permeabilize mitochondrial membranes

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G significantly accelerated hydroperoxide-induced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.     

Avicin D,a Plant Triterpenoid,Induces Cell Apoptosis by Recruitment of Fas and Downstream Signaling Molecules into Lipid Rafts

Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-β-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.     

Avicins, a novel plant-derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane

Avicins are pro-apoptotic, anti-inflammatory molecules with antioxidant effects both in vitro and in vivo. Based on their ability to perturb mitochondrial functions and initiate apoptosis in tumor cells, we chose to study the bioenergetic effects of avicins on tumor cell mitochondria. Avicin-treated Jurkat cells, showed a decrease in the levels of cellular ATP as well as the rate of oxygen consumption. These effects on cellular metabolism appear to be a result of avicin's actions on the outer mitochondrial membrane (OMM). We speculate that avicins might initially inhibit the exchange of metabolites across the OMM leading to its subsequent permeabilization to cytochrome c. This speculation is supported by biophysical studies using lipid bilayers, which suggest that upstream of these effects, avicins target and close the voltage dependent anion channel (VDAC). Closure of VDAC would lead to an overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization of the OMM and release of cyt-c. Avicins therefore not only represent a novel pharmacological tool for treatment of cancers, but also highlight the influence ancient plant metabolites could have on human health.     

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the “osmotic protection” method with 100 mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.     

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.     

Metabolomics of plant saponins: bioprospecting triterpene glycoside diversity with respect to mammalian cell targets

One of the goals of cancer chemotherapy and prevention is the discovery of compounds that are relatively selective to tumor cells and, therefore, have reduced effects on normal cell growth. In previously published studies, it was shown that certain triterpene saponins (called avicins) from a desert tree, Acacia victoriae, are selectively toxic to tumor cells at very low doses (IC(50): 0.2 microg/mL for Jurkat cells). To extend this research to human clinical studies, we needed to find a reliable supply of avicins and have developed a transformed "hairy root" culture as a means of biomass production. Protocols were optimized for A. victoriae micropropagation; after a boiling water treatment, A. victoriae seeds were maintained under in vitro conditions on defined media. Embryo-axis explants from shoot tips were removed and infected with Agrobacterium rhizogenes Conn (R 1000) for hairy root induction. Plasmid integration was confirmed by PCR analysis with a primer set for a segment of the rol B gene. Culture conditions have been optimized for root biomass production, and various inducers have been investigated for enhancement of avicin production. Hairy root cultures were compared with intact pod tissue from field-grown sources for avicin content following partial purification of triterpene glycosides and HPLC separation of the secondary metabolites. From bioassays of the collected HPLC fractions, we have identified putative triterpene "metabolic clusters" with enhanced activity against tumor cells. This now provides a system for both production of clinical trial lots of active samples, but also a means to correlate structure of individual triterpene glycosides with specific cellular target activity in mammalian cells.     

Mitochondria as integrators of information in an early-evolving animal: insights from a triterpenoid metabolite

Mitochondria have the capacity to integrate environmental signals and, in animals with active stem cell populations, trigger responses in terms of growth and growth form. Colonial hydroids, which consists of feeding polyps connected by tube-like stolons, were treated with avicis, triterpenoid electrophiles whose anti-cancer properties in human cells are mediated in part by mitochondria. In treated hydroids, both oxygen uptake and mitochondrial reactive oxygen species were diminished relative to controls, similar to that observed in human cells exposed to avicins. While untreated colonies exhibit more stolon branches and connections in the centre of the colony than at the periphery, treated colonies exhibit the opposite: fewer stolon branches in the centre of the colony than at the periphery. The resulting growth form suggest an inversion of the normal pattern of colony development mediated by mitochondrial and redox-related perturbations. An as-yet-uncharacterized gradient within the colony may determine the ultimate phenotypic effects of avicin perturbation.     

Proapoptotic triterpene electrophiles (avicins) form channels in membranes: cholesterol dependence

Avicins, a family of triterpenoid saponins from Acacia victoriae, can regulate the innate stress response in human cells. Their ability to induce apoptosis in transformed cells makes them potential anticancer agents. We report that avicins can form channels in membranes. The conductance reached a steady state after each addition, indicating a dynamic equilibrium between avicin in solution and in the membrane. The high power dependence (up to 10) of the membrane conductance on the avicin concentration indicates the formation of multimeric channels, consistent with the estimated pore radius of 1.1 nm. This radius is too small to allow protein flux across the mitochondrial outer membrane, a process known to initiate apoptosis. Channel formation is lost when avicin's amphipathic side chain is removed, implicating this as the channel-forming region. A small difference in this side chain results in strong cholesterol dependence of channel formation in avicin G that is not found in avicin D. In neutral membranes, avicin channels are nonselective, but negatively-charged lipids confer cation selectivity (5:1, K(+):Cl(-)), indicating that phospholipids form part of the permeation pathway. Avicin channels in the mitochondrial outer membrane may favor apoptosis by altering the potential across this membrane and the intermembrane space pH.