Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress.

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

Tobacco hairy root's peroxidases are rhythmically controlled by phenol exposure.

Plants like almost all living organisms, have developed a biological clock or circadian clock (CC) capable of synchronizing and adjusting various metabolic and physiological processes at certain times of the day and in a period of 24 h. This endogenous timekeeping is able to predict the environmental changes providing adaptive advantages against stressful conditions. Therefore, the aim of this work was to analyze the possible link between metabolism of xenobiotic compounds (MXC) and the CC. Synchronized Nicotiana tabacum hairy roots (HRs) were used as a validated plant model system, and peroxidases (PODs), key enzymes of the phase I in the MCX, were evaluated after phenol treatment. Two POD genes were selected and their temporal expression profiles as well as the total POD activity were analyzed in order to find circadian oscillations either under control conditions or phenol treatment. It was demonstrated that these PODs genes showed oscillatory profiles with an ultradian period (period length shorter than the circadian period), and preserving the same phases and expression peaks still under phenol treatment. The total PODs activity showed also a marked oscillatory behavior mainly in phenol-treated HRs with the highest levels at ZT23. Untreated HRs showed decrease and increase in the intensity of some basic isoforms at light and dark phase, respectively, while in phenol- treated HRs, an increase in the intensity of almost all isoforms was observed, mainly during the dark phase, being coincident with the high PODs activity detected at ZT23. The periodic analysis determined an ultradian period either in total POD activity or in the POD activity of isoform VI, being 18.7 and 15.3 h, respectively. Curiously, in phenol treated HRs, the period length of total POD activity was longer than in untreated HRs, suggesting that phenol could induce a marked oscillatory behavior in the POD activity with better performance during the dark phase, which explain the higher phenol removal efficiencies at ZT23. These findings showed novel information about the performance of PODs, which would be rhythmically controlled at biochemical level, by phenol exposure.

Temporal regulation of tumor growth in nocturnal mammals: In vivo studies and chemotherapeutical potential.

Tumors of the nervous system including glioblastoma multiforme (GBM) are the most frequent and aggressive form of brain tumors; however, little is known about the impact of the circadian timing system on the formation, growth, and treatment of these tumors. We investigated day/night differences in tumor growth after injection of A530 glioma cells isolated from malignant peripheral nerve sheath tumor (MPNSTs) of NPcis (Trp53+/- ; Nf1+/- ) mice. Synchronized A530 cell cultures expressing typical glial markers were injected at the beginning of the day or night into the sciatic nerve zone of C57BL/6 mice subject to a 12:12 hours light/dark (LD) cycle or after being released to constant darkness (DD). Tumors generated in animals injected early at night in the LD cycle or in DD showed higher growth rates than in animals injected diurnally. No differences were found when animals were injected at the same time with cultures synchronized 12 hours apart. Similar experiments performed with B16 melanoma cells showed higher tumor growth rates in animals injected at the beginning of the night compared to those injected in the daytime. A higher tumor growth rate than that in controls was observed when mice were injected with knocked-down clock gene Bmal1 cells. Finally, when we compared day/night administration of different doses of the proteasome inhibitor Bortezomib (0.5-1.5 mg/kg) in tumor-bearing animals, we found that low-dose chemotherapy displayed higher efficacy when administered at night. Results suggest the existence of a precise temporal control of tumor growth and of drug efficacy in which the host state and susceptibility are critical.

Impact of phenol on the glycerophospholipid turnover and potential role of circadian clock in the plant response against this pollutant in tobacco hairy roots.

Glycerophospholipids (GPLs) from cell membranes (CM) are a proper source for the synthesis of lipid messengers able to activate signal pathways that will define the plant survival under changing and stressful environmental conditions. Little is known about how GPLs metabolism (GPLsM) is regulated and the effects of phenol treatment on GPLs composition. In this work, we studied the effects of phenol both on GPLs turnover and on the expression of GPLsM-related genes potentially regulated by the circadian clock, using tobacco hairy root cultures (HRC). Phenol decreased the total PC levels and increased PE, PG and CL levels in the dark phase. Different molecular species of PC and PE showed the same trend than the total PC and PE upon phenol treatment. Besides, significant differences in the expression of all studied genes related to GPLsM were found. NtCCT2 expression was affected at all analyzed times while NtPECT1 and NtAAPT1 showed similar expression patterns. NtCDS1, NtPGPS2 and NtCLS genes showed significant and differential expression profiles both in untreated and treated HRC. PECT1 and NtPGPS2 genes seem to conserve a circadian expression profile mainly in untreated HRC. However, phenol was able to modify the GPLs composition and the expression of genes related to GPLs synthesis. The GPLs modification could be explained by the up-regulation of NtPECT1, NtAAPT1 and NtCLS genes during the dark phase, suggesting for being a crucial moment for HRC to trigger an adaptive response against this organic pollutant.

Catanionic nanocarriers as a potential vehicle for insulin delivery.

Diabetes is a disease that affects millions of people in the World, constituting a global problem. Patients are administered insulin subcutaneous injections, resulting in high costs and frequent infections in the injection site. A possible solution to this problem may be the use of nanotechnology. Nanotransporters can act as specific release systems able to overcome the current limitations to drug delivery. Liposomes and vesicles can deliver drugs directly and efficiently to the site of action, decreasing toxicity and adverse effects. In previous studies, we demonstrated the biocompatibility and safety of catanionic benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHD-AOT) vesicles using both in vitro and in vivo tests. Thus, the aims of this work were to evaluate the ability of the BHD-AOT vesicles to encapsulate insulin; to analyze the structural properties and stability of the system, vesicle-Insulin (VIn), at different pH conditions; and to study the ability of VIn to decrease the glycemia in miceby different administration routes. Our results showed that 2 and 5 mg mL-1 of vesicles were able to encapsulate about 55 % and 73 % of insulin, respectively. The system VIn showed a significant increase in size from 120 to 350 nm, changes in the surface zeta potential value, and high stability to different pH conditions. A significant decrease of the glycemia after VIn administration was demonstrated in in vivo assays, including the oral route. Our results reveal that BHD-AOT vesicles may be an appropriate system to encapsulate and protect insulin, and may be a potential system to be administrated in different ways as an alternative strategy to conventional therapy.

Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy.

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

Identification and characterization of key circadian clock genes of tobacco hairy roots: putative regulatory role in xenobiotic metabolism.

The circadian clock is an endogenous system that allows organisms to daily adapt and optimize their physiology and metabolism. We studied the key circadian clock gene (CCG) orthologs in Nicotiana tabacum seedlings and in hairy root cultures (HRC). Putative genes involved in the metabolism of xenobiotic compounds (MXC) were selected and their expression profiles were also analyzed. Seedlings and HRC displayed similar diurnal variations in the expression profiles for the CCG examined under control conditions (CC). MXC-related genes also showed daily fluctuations with specific peaks of expression. However, when HRC were under phenol treatment (PT), the expression patterns of the clock and MXC-related genes were significantly affected. In 2-week-old HRC, PT downregulated the expression of NtLHY, NtTOC1, and NtPRR9 while NtFKF1 and NtGI genes were upregulated by phenol. In 3-week-old HRC, PT also downregulated the expression of all CCG analyzed and NtTOC1 was the most affected. Following PT, the expression of the MXC-related genes was upregulated or displayed an anti-phasic expression profile compared to the expression under CC. Our studies thus provide a glimpse of the circadian expression of clock genes in tobacco and the use of HRC as a convenient system to study plant responses to xenobiotic stresses.

Phytoremediation of phenol using Vicia sativa L. plants and its antioxidative response.

Common vetch (Vicia sativa L.) is a legume species with an extensive agricultural use. However, the phytoremediation potentiality of this species has not been sufficiently explored because little is known about its resistance to inorganic and organic pollutants. In the present work, phenol tolerance of common vetch was assayed at different stages of growth. Germination index and germination rate decreased only at high phenol concentrations (250 and 500 mg L(-1)), whereas 30-day-old plants were able to tolerate this pollutant, with high removal efficiencies. The activities of antioxidative enzymes, such as peroxidase (POD) and ascorbate peroxidase, increased significantly with the highest phenol concentration, whereas superoxide dismutase activity, malondialdehyde, and H(2)O(2) levels remained unaltered. Besides, an increase in two basic isoforms of POD was observed in plants treated with phenol. The results suggested that common vetch has an efficient protection mechanism against phenol-induced oxidative damage. Moreover, it could tolerate and remove high phenol concentrations, avoiding serious phytotoxic effects. Thus, V. sativa could be considered an interesting tool in the field of phytoremediation.

Antioxidant response of tobacco (Nicotiana tabacum) hairy roots after phenol treatment.

Phenol is released to the environment from a wide variety of industrial effluents and it causes severe problems to human health and ecosystem. In the present study, we determined that Nicotiana tabacum hairy roots (HRs) double transgenic (DT) for two peroxidase genes (tpx1 and tpx2) showed higher phenol removal efficiency than wild type (WT) HRs after 120 h of phenol treatment at the expense of endogenous H(2)O(2). Besides, to determine whether phenol could induce oxidative stress on tobacco HRs, we analyzed the antioxidant response, superoxide anion (O(2)(-)) localization and malondialdehyde (MDA) levels. Both HRs treated with phenol, showed significant increases in peroxidase (PX) activity mainly at the end of the assay (120 h) being PX activity from transgenic HRs 40% higher than that of WT HRs. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities showed significant increases from 24 to 120 h of phenol treatment. PX, SOD and APX isoforms were also analyzed and slight changes were observed only in PX patterns. Both HRs showed significant differences in total glutathione (TGSH) content during treatment, being higher in DT HRs than in WT HRs. At the end of the assay, a greater accumulation of O(2)(-) in different root zones was observed in WT and DT HRs. Moreover, phenol was able to increase the MDA levels in WT HRs from 48 to 120 h of the treatment, but no significant changes were observed in DT HRs. Results suggest that under these experimental conditions, DT HRs would be more tolerant to phenol than WT HRs.

Establishment of transgenic tobacco hairy roots expressing basic peroxidases and its application for phenol removal.

Transgenic hairy root (HR) systems constitute an interesting alternative to improve the efficiency of phytoremediation process. Since peroxidases (Px) have been associated with phenolic compounds removal, in the present work, transgenic tobacco HR, which expressed basic Px genes from tomato (tpx1 and tpx2), were established and assayed for phenol removal. Tobacco HR clones were obtained, including those transgenic for TPX1 or TPX2, those double transgenic (DT) for both Px and the corresponding controls. Based on growth index, the presence of rol C sequence, tpx1 and/or tpx2 genes and the coded proteins, as well as Px activity determinations, we selected 10 tobacco HR clones for phenol removal assays. The removal efficiencies were high for all the HR, although, some transgenic HR showed significantly higher removal efficiencies compared with controls. The results demonstrate that TPX1 is involved in phenol removal not only when it was overexpressed in tomato, but also when it was expressed in other plant, such as tobacco. The higher efficiency of TPX2 transgenic HR showed that this Px also participates in the process. The contribution of other mechanisms (adsorption, H2O2 independent enzymatic processes) could be considered depreciable, which establishes the great implication of Px in phenol removal.