Iron Uptake in Ustilago maydis: Tracking the Iron Path

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.     

Purification and Characterization of Laccase from Chaetomium thermophilium and Its Role in Humification

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.     

Meiotic Recombination and DNA Synthesis in a New Cell Cycle Mutant of SACCHAROMYCES CEREVISIAE

Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.     

Fe nutrition demand and utilization by the green alga Dunaliella bardawil

The Fe nutritional demands, requirements and mechanisms of uptake by Dunaliella bardawil as well as potential Fe sources were studied. A comparison between Fe uptake from bacterial siderophores and from synthetic ferric chelates revealed algal growth response and chlorophyll synthesis to increasing concentrations and availability at a range of 0.01 μM–5 μM, as well as differences in efficiency. Furthermore, chloroplast ultrastructure, as observed by TEM, was affected by Fe deficiency, as was chlorophyll content. Ferric reduction is involved in the Fe uptake mechanism of Fe-stressed D. bardawil. Nutrient solution with controlled levels of free Fe²⁺ as well as spectrophotometric assays were used to measure Fe³⁺ reduction. This study shows that D. bardawil utilizes Fe³⁺ via a reduction mechanism, similar to that of strategy-I higher plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Drug-related thrombosis in hematologic malignancies

Cancer patients are at increased risk for thrombosis. Among the predisposing factors for the hemostatic imbalance, drugs have a definite role. Induction of thrombosis by drugs involves a variety of mechanisms: Enhancement of procoagulant activity, reduction in anticoagulants synthesis, stimulation of platelet aggregation and endothelial damage. L-asparaginase is associated with thrombotic events, mainly in the venous system. Supportive therapy with fresh frozen plasma is probably insufficient and heparin needs further evaluation. Venous thromboembolism has recently emerged following thalidomide use particularly in combination chemotherapy. The hematopoietic growth factors granulocyte colony-stimulating factor, macrophage-granulocyte colony-stimulating factor and erythropoietin have also been implicated in venous as well as in arterial thrombotic events. Numerous drugs are associated with thrombotic microangiopathy i.e., cyclosporine A, tacrolimus, cisplatin, bleomycin, gemcitabine. The clinical presentation, pathological mechanisms and therapeutic modalities are discussed.     

Control of in vitro rooting and plant development in Corymbia maculata by silver nitrate, silver thiosulfate and thiosulfate ion

Plant regeneration and transformation in vitro is often improved by adding silver ion (Ag⁺) to the culture media as AgNO₃ or silver thiosulfate (STS). Ag⁺ reacts with substances to form insoluble precipitates, while thiosulfate (S₂O₃ ²⁻) interferes with these reactions. We studied the implications of silver precipitation and S₂O₃ ²⁻ in the medium for culture development by (1) examining formation of Ag⁺ precipitates from AgNO₃ versus STS in agar gels and their possible dependence on agar type; (2) comparing Corymbia maculata culture responses to AgNO₃ and STS and determining which better suits control of culture development; (3) clarifying whether STS-dependent alterations in culture development are due to Ag⁺ alone or also to a separate influence of S₂O₃ ²⁻. Silver precipitates appeared in aqueous gels of four agar brands supplemented with AgNO₃, but not in Phytagel^™, which remained transparent. No precipitation was observed in gels with STS. Indole-3-butyric acid (IBA)-mediated adventitious root induction and shoot growth were higher in C. maculata shoot tips cultured on gels with STS versus AgNO₃ (6–25 μM Ag⁺). IBA-treated shoot tips exhibited enhanced adventitious root regeneration, accelerated root elongation, increased frequency of lateral root formation, and stimulated shoot growth mediated by 100–250 μM sodium thiosulfate (Na₂S₂O₃) in medium without Ag⁺. The potency of S₂O₃ ²⁻ in facilitating culture development has never been recognized. It is inferred that superiority of STS in stimulating multiple responses of C. maculata culture results from sustained biological activity of Ag⁺ through prevention of its precipitation, and from impact of S₂O₃ ²⁻ on cell differentiation and growth.     

Regulation of cancer aggressive features in melanoma cells by microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.     

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.