Participation of actin filaments, myosin and phosphatidylinositol 3-kinase in the formation and polarisation of tetraspore germ tube of Gelidium floridanum (Rhodophyta, Florideophyceae).

This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3-kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum. After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6 h. In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F-actin. In the presence of cytochalasin B, an inhibitor of F-actin, latrunculin B, an inhibitor of G-actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical-shaped chloroplasts were observed in the central region with a few F-actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation. It was concluded that F-actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F-actin. Thus, F-actin regulates the polarisation and germination processes of tetraspores of G. floridanum.

Tolerance of Ulothrix sp. LAFIC 010 (Chlorophyta) against high concentration of metals from acid mine drainage.

The production of acid mine drainage of (AMD) is one of the main phenomena responsible for much of the degradation of water and soil resources. Organisms present at sites contaminated by AMD can have the potential to bioaccumulate heavy metals, stimulating their application in bioremediation processes. Ulothrix sp. LAFIC 010 was identified among the species of algae isolated from water contaminated by AMD in the region of Sideropólis (Brazil). The present study evaluated its tolerance and bioaccumulation potential related to zinc, manganese and nickel. Experiments were performed to see the effects of different concentrations of Zn, Mn and Ni (individually and in combination) on the physiological performance of the alga. The results showed that only the cultures submitted to concentrations above 0.55 mM Zn showed a decrease in growth rate and damage to physiological processes. There was no observed effect of Mn and Ni on Ulothrix sp. LAFIC 010 physiology, even with an 8-fold increase in concentrations of these metals in the medium. In cultures with combined metals, only the treatments with the highest concentrations of Zn presented reduced growth, regardless of the presence of other metals. Additionally, we observed that Mn and Ni did not decrease the toxic effect of Zn. Mn accumulation was indicated in the cell wall and Ni in the vacuole. Our results suggest that the distribution of this alga in contaminated medium is not affected by the concentration of Ni and Mn, at least under the pH that was evaluated. We conclude that Ulothrix sp. LAFIC 010 tolerates and grows under conditions with higher metal concentrations than previously reported for AMD.

Photosynthetic and ultrastructural responses of Ulva australis to Zn stress.

This research evaluated the effect of zinc (Zn) on the ultrastructure and the photosynthetic efficiency of a common green alga. Ulva australis was grown in the laboratory for 7days under a range of different Zn concentrations (0, 25, 50 and 100µgL-1). Growth rate (Gr), photosynthetic efficiency (Fv/Fm and ETRmax), photosynthetic pigments, and metal accumulation were measured. Samples of 1mm length were taken to analyse the effect of Zn on the ultrastructure using transmission electron microscopy (TEM) and cytochemical responses (TB-O and PAS) were evaluated by light microscopy (LM). There were no significant differences in the growth rate, Fv/Fm, ETRmax and the photosynthetic pigments chlorophyll a, chlorophyll b and carotenoids (p>0.05) after 7days of Zn exposure. However, TEM revealed cytoplasm retraction, compression of cellulose fibrils, dissembled thylakoids and electron-dense bodies suggesting ultrastructural impacts from metal exposure and accumulation. Cytological analysis demonstrated that Zn affected U. australis cells at the three concentrations tested. The main effect was cytoplasm retraction and a decrease on the amount of starch granules, following exposure at 25µgL-1 and 50µgL-1 of Zn. We conclude that concentrations of Zn assessed in U. australis in this research has a short-term cellular effect as revealed by TEM and cytological analysis, demonstrating the importance of measuring a broad suite of endpoints to better understand species responses to environmentally relevant concentrations of Zn. However, U. australis was able to physiologically tolerate adverse conditions, since there was no effect on the photosynthetic performance and growth.

Fine-tuning transmission electron microscopy methods to evaluate the cellular architecture of Ulvacean seaweeds (Chlorophyta).

Chemical fixation is a critical step in the analysis of the ultrastructure of seaweeds because the wrong approach can compromise the ability to distinguish fine-scale cellular composition. Fixation agents, fixation time and type of tissue are important factors to consider for transmission electron microscopy (TEM), and not every protocol is suitable for all cell types. We evaluated a range of fixation agents, post-fixation time and dehydration solutions to determine a TEM protocol for seaweeds in the Family Ulvaceae. We assessed Ulva lactuca using 5 protocols. The level of preservation obtained differed markedly between fixation methods The best result was obtained by fixing the sample with 2.5% glutaraldehyde, 0.05M sodium cacodylate buffer and 2% paraformaldehyde overnight, and 8h post-fixation in 1% in osmium tetroxide 1%. This approach and fixation time ensured that the membranes, especially the thylakoid membranes of chloroplasts, remained intact. Ethanol is recommended for dehydration as the use of acetone for dehydration resulted in the collapse of cellular membranes. This new protocol will ensure the ultrastructure of Ulvacean seaweeds can be clearly ascertained in the future.

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential.

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.