Magda Tušek Žnidarič

Arsenic Trioxide (ATO) Influences the Gene Expression of Metallothioneins in Human Glioblastoma Cells

Arsenic trioxide (As2O3; ATO, TRISENOX®) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6–7xa0μM arsenic (equivalent to 0.3, 1 and 3–3.5xa0μM As2O3) for 12, 24 or 48xa0h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and Taqman® gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.

Structural characterization of liposomes made of diether archaeal lipids and dipalmitoyl-L-α-phosphatidylcholine.

The physicochemical properties of binary lipid mixtures of diether C(25,25) lipids and dipalmitoyl-L-α-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol%C(25,25) lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.

Clinical and Structural Features of Sperm Head Vacuoles in Men Included in the In Vitro Fertilization Programme

The human sperm head vacuoles and their role in male infertility are still poorly understood. The aim of this study was to identify the clinical and ultrastructural features of human sperm head vacuoles in men included in the in vitro fertilization programme: men with normal (normozoospermia) and impaired sperm morphology (teratozoospermia). The sperm samples were observed under 6000-time magnification using motile sperm organelle morphology examination (MSOME). The proportion of sperm with head vacuoles was evaluated and related to the outcome of in vitro fertilization. The sperm of men with impaired sperm morphology was characterized by a higher proportion of sperm head vacuoles. The sperm head vacuoles were related to impaired semen quality (sperm concentration, motility, and morphology) but were not influenced by male factors (semen volume, height, age, weight, or body mass index). Moreover, sperm head vacuoles were related to impaired fertilization rate merely after classical in vitro fertilization (IVF), while there was no relation to pregnancy. In a subgroup of men, the sperm was fixed and observed by transmission electron microscopy (TEM). The ultrastructural study revealed that sperm head vacuoles are large nuclear indentations of various sizes and positions, packed with membranous material organized in membrane whorls (MW).

Cd, Cu, Zn, Se, and metallothioneins in two amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura).

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85xa0μg/L) for up to 40xa0days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1xa0μg/g in kidneys and gills (0.64–0.95 and 0.52–0.76; nu2009=u20094), whereas the levels stayed below 0.5 in liver (0.14–0.29; nu2009=u20094) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn–thioneins in liver and Zn,Cu–thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd–thionein and in pellet extract of kidneys as Zn,Cu,Cd–thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56–5.0xa0μg/g >0.03–0.72xa0μg/g; nu2009=u200911, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all ru2009>u20090.80; αu2009=u20090.05, nu2009=u20095), and between Cd and Se in kidney (ru2009=u20090.76; nu2009=u20095) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd–thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.

Annual cambial rhythm in Pinus halepensis and Pinus sylvestris as indicator for climate adaptation

To understand better the adaptation strategies of intra-annual radial growth in Pinus halepensis and Pinus sylvestris to local environmental conditions, we examined the seasonal rhythm of cambial activity and cell differentiation at tissue and cellular levels. Two contrasting sites differing in temperature and amount of precipitation were selected for each species, one typical for their growth and the other represented border climatic conditions, where the two species coexisted. Mature P. halepensis trees from Mediterranean (Spain) and sub-Mediterranean (Slovenia) sites, and P. sylvestris from sub-Mediterranean (Slovenia) and temperate (Slovenia) sites were selected. Repeated sampling was performed throughout the year and samples were prepared for examination with light and transmission electron microscopes. We hypothesized that cambial rhythm in trees growing at the sub-Mediterranean site where the two species co-exist will be similar as at typical sites for their growth. Cambium in P. halepensis at the Mediterranean site was active throughout the year and was never truly dormant, whereas at the sub-Mediterranean site it appeared to be dormant during the winter months. In contrast, cambium in P. sylvestris was clearly dormant at both sub-Mediterranean and temperate sites, although the dormant period seemed to be significantly longer at the temperate site. Thus, the hypothesis was only partly confirmed. Different cambial and cell differentiation rhythms of the two species at the site where both species co-exist and typical sites for their growth indicate their high but different adaptation strategies in terms of adjustment of radial growth to environmental heterogeneity, crucial for long-term tree performance and survival.

1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene

Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.

Metal binding of metallothioneins in human astrocytomas (U87 MG, IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1xa0μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24xa0hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48xa0hours with exposure to different concentrations of CdCl2 (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.

Metal(loid)s Accumulation and Interactions in Tetrahymena thermophila

ABSTRACT Metal(loid)s accumulation and interactions were studied in Tetrahymena thermophila after 24 h exposure to 100 µM CuI, 20 µM CdII, 1 µM of AgI, HgII, PbII, AsIII, AsV, SbIII, SbV, SeIV, or SeVI, and once to Cu deprivation. More than 80% of elements adopted to the cells (except Ag) were found in water-insoluble form. Cu exposure led in co-accumulation of Cu with zinc, iron and selenium. In cell lysates the studied elements, particularly Cd, Hg, Pb, and Sb, were more or less associated with metallothioneins. Most exposures had a negative impact on cellular consumption and content of essential metals Cu, Zn, and Fe.

Detection of Phytoplasmas Associated to Grapevine Yellows Diseases in Research and Diagnostics

Research into grapevine yellows diseases and their control relies on detection and identification of the phytoplasmas associated with them. Detection methods for phytoplasmas can be divided into four main categories: biological tests, microscopy techniques, and immunological and molecular approaches. The suitability of each of these methods for different studies is discussed in this chapter, along with their advantages and disadvantages. Among these methods, PCR-based assays in particular are routinely used in diagnostic laboratories because of their high sensitivity and potential to be automated for high-throughput testing. Recently, isothermal amplification methods have been developed for rapid on-site phytoplasma diagnostics, such as loop-mediated isothermal amplification assays. The development of any diagnostic assay requires thorough validation to ensure its sensitivity, specificity, repeatability, and reproducibility and that the assay is fit for purpose. In addition, for validated detection, measures to reduce the uncertainty of tests that are carried out need to be implemented through the whole diagnostic process, which must therefore also be robust.

Crustacean Cuticle: Synthesis and Remodeling of a Dynamic Extracellular Matrix During molt Cycle

Cuticle is mostly considered as a nonliving part of crustacean body functioning as a mechanical and permeability barrier in different environments. In this contribution we want to expose the idea that cuticle is a living part of the animal body perforated by numerous pore channels with cytoplasmic extensions delivering various components to the newly synthesized extracellular matrix which is constantly remodeled during molt cycle. Terrestrial isopod crustaceans developed an elaborate cuticular matrix with different degree of calcification as an adaptation to various land habitats, ranging from sea shores to deserts.