Gordana Timotijevic

Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast

· Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast. · A combined approach was performed including analyses of apoplastic Fe, reduction-based Fe acquisition and Fe-mobilizing compounds in roots along with the expression of related genes. · Si-treated plants accumulated higher concentrations of root apoplastic Fe, which rapidly decreased when Fe was withheld from the nutrient solution. Under Fe-deficient conditions, Si also increased the accumulation of Fe-mobilizing compounds in roots. Si supply stimulated root activity of Fe acquisition at the early stage of Fe deficiency stress through regulation of gene expression levels of proteins involved in Fe acquisition. However, when the period of Fe deprivation was extended, these reactions further decreased as a consequence of Si-induced enhancement of the Fe status of the plants. · This work provides new evidence for the beneficial role of Si in plant nutrition and clearly indicates that Si-mediated alleviation of Fe deficiency includes an increase of the apoplastic Fe pool in roots and an enhancement of Fe acquisition.

Expression analysis of buckwheat (Fagopyrum esculentum Moench) Metallothionein-like gene (MT3) under different stress and physiological conditions

The buckwheat metallothionein-like (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. MT3 mRNAs were detected from the early stage of seed development to the end of maturation, reaching the highest level during the mid-maturation stage. High MT3 mRNA level was noticed for both green and senescent leaves. The influence of raising Cu ion concentrations on MT3 gene expression was studied only in leaves, while the effect of Zn ions was analyzed through seed development as well. It was found that Cu and Zn ions had stimulatory effects on expression in leaves. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions, while after this stage, influence of Zn ions was not detected. After H2O2/NaCl treatment, MT3 mRNA level was decreased in green leaves, contrary to senescent leaves where expression levels remained unchanged. H2O2 treatment caused the increase of MT3 mRNA levels in the mid-maturation stage of seed development. NaCl had no effect on expression levels in seeds. According to obtained results, proposed functions in different plant organs regarding oxidative stress and metal homeostasis are discussed.

Astrocytes in the tempest of multiple sclerosis

Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity‐related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes.

Macrophage migration inhibitory factor deficiency protects pancreatic islets from palmitic acid-induced apoptosis

As a result of chronic exposure to high levels of free fatty acids, glucose and inflammatory mediators β‐cell apoptosis occurs at the end stage of obesity‐associated type 2 diabetes (T2D). One potentially deleterious molecule for β‐cell function associated with T2D and obesity in humans is macrophage migration inhibitory factor (MIF). Therefore, the aim of this study was to explore MIF expression in vivo during development of obesity and insulin resistance in high‐fat diet (HFD)‐fed C57BL/6 mice and whether MIF inhibition could affect β‐cell apoptosis and dysfunction induced by palmitic acid (PA) in vitro. Indeed, increase in systemic and locally produced MIF correlated well with the weight gain, triglyceride upregulation, glucose intolerance and insulin resistance, which developed in HFD‐fed mice. In in vitro settings PA dose‐dependently induced MIF secretion before apoptosis development in islets. Further, mif gene deletion, mRNA silencing or protein inhibition rescued β‐cells from PA‐induced apoptosis as measured by MTT assay and histone‐DNA enzyme linked immuno sorbent assay. Protection from induced apoptosis was mediated by altered activation of caspase pathway and correlated with changes in the level of Bcl‐2 family members. Further, MIF inhibition conveyed a significant resistance to PA‐induced downregulation of insulin and PDX‐1 expression and ATP content. However, β‐cell function was not entirely preserved in the absence of MIF judging by low glucose oxidation and depolarized mitochondrial membrane. In conclusion, the observed considerable preservation of β‐cells from nutrient‐induced apoptosis might implicate MIF as a potential therapeutic target in the later stage of obesity‐associated T2D.

Multiple antimelanoma potential of dry olive leaf extract.

Various constituents of the olive tree (Olea europaea) have been traditionally used in the treatment of infection, inflammation, prevention of chronic diseases, cardiovascular disorders and cancer. The anticancer potential of dry olive leaf extract (DOLE) represents the net effect of multilevel interactions between different biologically active compounds from the extract, cancer cells and conventional therapy. In this context, it was of primary interest to evaluate the influence of DOLE on progression of the highly malignant, immuno‐ and chemoresistant type of skin cancer—melanoma. DOLE significantly inhibited proliferation and subsequently restricted clonogenicity of the B16 mouse melanoma cell line in vitro. Moreover, late phase tumor treatment with DOLE significantly reduced tumor volume in a syngeneic strain of mice. DOLE‐treated B16 cells were blocked in the G0/G1 phase of the cell cycle, underwent early apoptosis and died by late necrosis. At the molecular level, the dying process started as caspase dependent, but finalized as caspase independent. In concordance, overexpression of antiapoptotic members of the Bcl‐2 family, Bcl‐2 and Bcl‐XL, and diminished expression of their natural antagonists, Bim and p53, were observed. Despite molecular suppression of the proapoptotic process, DOLE successfully promoted cell death mainly through disruption of cell membrane integrity and late caspase‐independent fragmentation of genetic material. Taken together, the results of this study indicate that DOLE possesses strong antimelanoma potential. When DOLE was applied in combination with different chemotherapeutics, various outcomes, including synergy and antagonism, were observed. This requires caution in the use of the extract as a supplementary antitumor therapeutic.

In vitro and in vivo anticancer action of Saquinavir-NO, a novel nitric oxide-derivative of the protease inhibitor saquinavir, on hormone resistant prostate cancer cells.

The NO-derivative of the HIV protease inhibitor saquinavir (Saq-NO) is a nontoxic variant of the parental drug with enhanced anticancer activity on several cell lines. However, it is still unclear whether the p53 status of the target cell might influence the sensitivity to Saq-NO. In this study we evaluated the in vitro and in vivo activity of Saq-NO on the p53-deficient hormone resistant prostate cancer PC-3 cells. We demonstrate that the absence of functional p53 is not essential for the capacity of Saq-NO to reduce prostate cancer cell growth. In contrast to its previously described cytostatic action in B16 and C6 cell lines, Saq-NO exerted cytotoxic effects in PC-3 cells leading to dominant induction of apoptosis and enhanced production of proapoptotic Bim. In addition, differently from saquinavir, Saq-NO restored TRAIL sensitivity that was correlated with increased expression of DR5 independent from ROS/RNS production and YY1 repression. NF-κB activation may be responsible of the Saq-NO induced DR5 expression. Moreover, Saq-NO but not saquinavir, exerted synergistic activity with conventional cytostatic therapy. In agreement with these in vitro studies, Saq-NO inhibited the in vivo growth of PC-3 cells xenotransplants to a greater extent than the parental compound. Taken together, these data indicate that Saq-NO possesses powerful and suitable in vitro and in vivo chemotherapeutic potential to be further studied as a novel drug for the treatment of prostate cancer in the clinical setting.

The antitumor properties of a nontoxic, nitric oxide–modified version of saquinavir are independent of Akt

Application of the HIV protease inhibitor saquinavir (Saq) to cancer chemotherapy is limited by its numerous side effects. To overcome this toxicity, we modified the original compound by covalently attaching a nitric oxide (NO) group. We compared the efficacy of the parental and NO-modified drugs in vitro and in vivo. The novel compound saquinavir-NO (Saq-NO) significantly reduced the viability of a wide spectrum of human and rodent tumor cell lines at significantly lower concentration than the unmodified drug. In contrast to Saq, Saq-NO had no effect on the viability of primary cells and drastically reduced B16 melanoma growth in syngeneic C57BL/6 mice. In addition, at the equivalent of the 100% lethal dose of Saq, Saq-NO treatment caused no apparent signs of toxicity. Saq-NO blocked the proliferation of C6 and B16 cells, up-regulated p53 expression, and promoted the differentiation of these two cell types into oligodendrocytes or Schwann-like cells, respectively. Although it has been well documented that Saq decreases tumor cell viability by inhibiting Akt, the anticancer properties of Saq-NO were completely independent of the phosphatidylinositol 3-kinase/Akt signaling pathway. Moreover, Saq-NO transiently up-regulated Akt phosphorylation, delivering a protective signal that could be relevant for primary cell protection and the absence of drug toxicity in vivo. It was unlikely that released NO was independently responsible for these drug effects because Saq-NO treatment increased intracellular and secreted NO levels only slightly. Rather, the chemical modification seems to have produced a qualitatively new chemical entity, which may have a unique mode of action against cancer cells.[Mol Cancer Ther 2009;8(5):1169–78]

Cytotoxic and immune-sensitizing properties of nitric oxide-modified saquinavir in iNOS-positive human melanoma cells†‡

We have recently shown that covalent attachment of the NO moiety to the HIV protease inhibitor Saquinavir (Saq) produced a qualitatively new chemical entity, named Saquinavir‐NO (Saq‐NO), with enhanced anticancer properties and reduced toxicity. In this study we evaluated the impact of Saq‐NO on the growth of A375 human melanoma cells, as a prototype of NO‐dependent cancer model. The novel compound strongly affected the in vitro and in vivo progression of A375 melanoma cell growth. The mechanism of antimelanoma action comprised dual drug activity—induction of apoptotic cell death and acquisition of melanoma cell responsiveness to TRAIL. Saq‐NO‐triggered apoptosis was dependent on transient AKT up‐regulation and reduced pERK and iNOS expression that were observed within the first 12 h of exposure to the drug. Thereafter, however, Saq‐NO up‐regulated both iNOS transcription and NO endogenous synthesis and sensitized A375 cells to TRAIL. Furthermore, reduced YY1 expression was observed after 24 h of Saq‐NO exposure, which correlated with increased expression of DR5. The biological relevance of this complex and powerful action of Saq‐NO was consistent with the marked drug‐induced inhibition of the growth of A375 xenotransplants in nude mice. J. Cell. Physiol. 226: 1803–1812, 2011.

Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum).

Metallothionein type 3 (MT3) expression has previously been detected in leaves, fruits, and developing somatic embryos in different plant species. However, specific tissular and cellular localization of MT3 transcripts have remained unidentified. In this study, in situ RNA-RNA analysis revealed buckwheat metallothionein type 3 (FeMT3) transcript localization in vascular elements, mesophyll and guard cells of leaves, vascular tissue of roots and throughout the whole embryo. Changes in FeMT3 mRNA levels in response to drought and oxidative stress, as well as ROS scavenging abilities of the FeMT3 protein in yeast were also detected, indicating possible involvement of FeMT3 in stress defense and ROS related cellular processes.

Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats.

Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.