Spatial distribution of cadmium in leaves of metal hyperaccumulating Thlaspi praecox using micro-PIXE

* Localization of cadmium (Cd) and other elements was studied in the leaves of the field-collected cadmium/zinc (Cd/Zn) hyperaccumulator Thlaspi praecox from an area polluted with heavy metals near a lead mine and smelter in Slovenia, using micro-PIXE (proton-induced X-ray emission). * The samples were prepared using cryofixation. Quantitative elemental maps and average concentrations in whole-leaf cross-sections and selected tissues were obtained. * Cd was preferentially localized in the lower epidermis (820 microg g(-1) DW), vascular bundles and upper epidermis, whereas about twice the lower concentrations were found in the mesophyll. * Taking into account the large volume of the mesophyll compared with the epidermis, the mesophyll is indicated as a relatively large pool of Cd, possibly involved in Cd detoxification/dilution at the tissue and cellular level.     

CD4+ T-cell-epitope escape mutant virus selected in vivo.

Mutations in viral genomes that affect T-cell-receptor recognition by CD8+ cytotoxic T lymphocytes have been shown to allow viral evasion from immune surveillance during persistent viral infections. Although CD4+ T-helper cells are crucially involved in the maintenance of effective cytotoxic T-lymphocyte and neutralizing-antibody responses, their role in viral clearance and therefore in imposing similar selective pressures on the virus is unclear. We show here that transgenic virus-specific CD4+ Tcells, transferred into mice persistently infected with lymphocytic choriomeningitis virus, select for T-helper epitope mutant viruses that are not recognized. Together with the observed antigenic variation of the same T-helper epitope during polyclonal CD4+ T-cell responses in infected pore-forming protein-deficient C57BL/6 mice, this finding indicates that viral escape from CD4+ T lymphocytes is a possible mechanism of virus persistence.     

Physiological responses to Cd and Zn in two Cd/Zn hyperaccumulating Thlaspi species

In a model hyperaccumulation study a Cd/Zn hyperaccumulator Thlaspi caerulescens accession Ganges and a recently reported Cd/Zn hyperaccumulator Thlaspi praecox grown in increasing Cd and Zn concentrations in the substrate and in field collected polluted soil were compared. Plant biomass, concentrations of Cd and Zn, total chlorophylls and anthocyanins, antioxidative stress parameters and activities of selected antioxidative enzymes were compared. Increasing Cd, but not Zn in the substrate resulted in the increase of biomass of roots and shoots of T. praecox and T. caerulescens. The two species hyperaccumulated Cd in the shoots to a similar extent, whereas T. caerulescens accumulated more Zn in the shoots than T. praecox. Cadmium amendment decreased total chlorophyll concentration and glutathione reductase activity, and increased non-protein thiols concentration only in T. praecox, suggesting that it is less tolerant to Cd than T. caerulescens. In the field-contaminated soil, T. caerulescens accumulated higher Cd concentrations; but as T. praecox produced higher biomass, both species have similar ability to extract Cd.     

UV-B radiation affects flavonoids and fungal colonisation in <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> and <Emphasis Type="Italic">F. tataricum</Emphasis>

In the present study, we have evaluated the effects of increased UV-B radiation that simulates 17% ozone depletion, on fungal colonisation and concentrations of rutin, catechin and quercetin in common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum). Induced root growth and reduced shoot:root ratios were seen in both of these buckwheat species after enhanced UV-B radiation. There was specific induction of shoot quercetin concentrations in UV-B-treated common buckwheat, whereas there were no specific responses for flavonoid metabolism in tartary buckwheat. Root colonisation with arbuscular mycorrhizal fungi significantly reduced catechin concentrations in common buckwheat roots, and induced rutin concentrations in tartary buckwheat, but did not affect shoot concentrations of the measured phenolics. Specific UV-B-related reductions in the density of microsclerotia were observed in tartary buckwheat, indicating a mechanism that potentially affects fungus-plant interactions. The data support the hypothesis that responses to enhanced UV-B radiation can be influenced by the plant pre-adaptation properties and related changes in flavonoid metabolism.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Farnesyl Pyrophosphate Inhibits Epithelialization and Wound Healing through the Glucocorticoid Receptor

Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.     

High incidence of arbuscular mycorrhizal fungi in rare and endangered wild grapevine

High levels of arbuscular mycorrhizal fungi colonisation of 90–100%, as well as high vesicular and arbuscular colonisation were found in the roots of the rare and endangered wild grapevine (Vitis vinifera subsp. sylvestris) along the Neretva River in southwestern Bosnia and Herzegovina, surpassing colonisation in region vineyads.     

Induction of Specific MicroRNAs Inhibits Cutaneous Wound Healing

Chronic nonhealing wounds, such as venous ulcers (VUs), are a widespread and serious medical problem with high morbidity and mortality. The molecular pathology of VUs remains poorly understood, impeding the development of effective treatment strategies. Using mRNA expression profiling of VUs biopsies and computational analysis, we identified a candidate set of microRNAs with lowered target gene expression. Among these candidates, miR-16, -20a, -21, -106a -130a, and -203 were confirmed to be aberrantly overexpressed in a cohort study of 10 VU patients by quantitative PCR and in situ hybridizations. These microRNAs were predicted to target multiple genes important for wound healing, including early growth response factor 3, vinculin, and leptin receptor (LepR). Overexpression of the top up-regulated miRNAs, miR-21 and miR-130a, in primary human keratinocytes down-regulated expression of the endogenous LepR and early growth response factor 3. The luciferase reporter assay verified LepR as a direct target for miR-21 and miR-130a. Both miR-21 and miR-130a delayed epithelialization in an acute human skin wound model. Furthermore, in vivo overexpression of miR-21 inhibited epithelialization and granulation tissue formation in a rat wound model. Our results identify a novel mechanism in which overexpression of specific set of microRNAs inhibits wound healing, resulting in new potential molecular markers and targets for therapeutic intervention.     

Binding and permeabilization of lipid bilayers by natural and synthetic 3-alkylpyridinium polymers

Naturally occurring 3-alkylpyridinium polymers from the marine sponge Reniera sarai are membrane-active compounds exerting a selective cytotoxicity towards non small cell lung cancer cells, and stable transfection of nucleated mammalian cells. In view of their possible use as chemotherapeutics and/or transfection tools, three poly-APS based synthetic compounds were tested on their activity using natural and artificial lipid membranes. The tested compounds were found to be very stable over a wide range of temperature, ionic strength, and pH, and to prefer the solid-ordered membrane state. Their membrane-damaging activity increases with the length of their alkyl chains and the degree of polymerization.