A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

Glutathione peroxidases in different stages of carcinogenesis

Cancer cells produce high amounts of reactive oxygen species (ROS) and evade apoptosis. Hydroperoxides support proliferation, invasion, migration and angiogenesis, but at higher levels induce apoptosis, thus being pro- and anti-carcinogenic. Accordingly, glutathione peroxidases (GPxs) regulating hydroperoxide levels might have dual roles too. GPx1, clearly an antioxidant enzyme, is down-regulated in many cancer cells. Its main role would be prevention of cancer initiation by ROS-mediated DNA damage. GPx2 is up-regulated in cancer cells. GPx1/GPx2 double knockout mice develop colitis and intestinal cancer. However, GPx2 knockdown cancer cells grow better in vitro and in vivo probably reflecting the physiological role of GPx2 in intestinal mucosa homeostasis. GPx2 counteracts COX-2 expression and PGE₂ production, which explains its potential to inhibit migration and invasion of cultured cancer cells. Overexpression of GPx3 inhibits tumor growth and metastasis. GPx4 is decreased in cancer tissues. GPx4-overexpressing cancer cells have low COX-2 activity and tumors derived therefrom are smaller than from control cells and do not metastasize. Collectively, GPxs prevent cancer initiation by removing hydroperoxides. GPx4 inhibits but GPx2 supports growth of established tumors. Metastasis, but also apoptosis, is inhibited by all GPxs. GPx-mediated regulation of COX/LOX activities may be relevant to early stages of inflammation-mediated carcinogenesis.     

Raman spectroscopy a promising technique for investigations of metallothioneins

Metallobiomolecules are highly elaborated coordination complexes, and their fundamental metal-ligand interactions are critical components of metalloprotein folding, assembly, stability, electrochemistry, and catalytic function. Herein, we have described the benefits in using Raman spectroscopy to define the metal-ion binding properties of MTs toward metal ions such as Zn(ii) and Cd(ii). In particular, this vibrational technique can shed light on the secondary structures eventually present in MTs and the ligands involved in metal coordination. The oxidation state of Cys residues and their participation in the metal chelation can be clearly defined, as well as the eventual involvement of His residues. With regards to exogenous metal ligands such as sulfide anions, their presence can be identified by some marker bands whose intensity is linearly correlated with sulfide/metal molar ratio. Finally, Raman can be also an useful tool for providing information on the favourite sites of the radical attack and radical-induced modification in protein folding. In conclusion, many advantages such as the capability of defining local regions in large complexes and detecting several structural features at the same time, the ability in supporting mechanisms, as well as the requirement of low sample amount, make to propose Raman spectroscopy, in coupling with analytical techniques such as atomic emission spectroscopy, gas chromatography, and circular dichroism, as one of the most promising experimental strategies in the research on structure-activity relationships in MTs.     

Pollen performance of Raphanus sativus (Brassicaceae) declines in response to elevated [CO2]

Although increases in atmospheric [CO₂] are known to affect plant physiology, growth and reproduction, understanding of these effects is limited because most studies of reproductive consequences focus solely on female function. Therefore, we examined the effects of CO₂ enrichment on male function in the annual Raphanus sativus. Pollen donors grown under elevated [CO₂] initially sired a higher proportion of seeds per fruit than ambient [CO₂]-grown plants when each was tested against two different standard competitors; however, by the end of the 5-month experiment, these pollen donors sired fewer seeds than ambient [CO₂]-grown plants and produced a lower proportion of viable pollen grains. The results of this experiment confirm that elevated [CO₂] can alter reproductive success. Additionally, the change in response to elevated [CO₂] over time varied among pollen donor families; thus, changes in [CO₂] could act as a selective force on this species.     

Brain Metabolism during Hallucination-Like Auditory Stimulation in Schizophrenia

Auditory verbal hallucinations (AVH) in schizophrenia are typically characterized by rich emotional content. Despite the prominent role of emotion in regulating normal perception, the neural interface between emotion-processing regions such as the amygdala and auditory regions involved in perception remains relatively unexplored in AVH. Here, we studied brain metabolism using FDG-PET in 9 remitted patients with schizophrenia that previously reported severe AVH during an acute psychotic episode and 8 matched healthy controls. Participants were scanned twice: (1) at rest and (2) during the perception of aversive auditory stimuli mimicking the content of AVH. Compared to controls, remitted patients showed an exaggerated response to the AVH-like stimuli in limbic and paralimbic regions, including the left amygdala. Furthermore, patients displayed abnormally strong connections between the amygdala and auditory regions of the cortex and thalamus, along with abnormally weak connections between the amygdala and medial prefrontal cortex. These results suggest that abnormal modulation of the auditory cortex by limbic-thalamic structures might be involved in the pathophysiology of AVH and may potentially account for the emotional features that characterize hallucinatory percepts in schizophrenia.     

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.     

Activation of the endocannabinoid system by organophosphorus nerve agents

Delta(9)-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana, has useful medicinal properties but also undesirable side effects. The brain receptor for THC, CB(1), is also activated by the endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG). Augmentation of endocannabinoid signaling by blockade of their metabolism may offer a more selective pharmacological approach compared with CB(1) agonists. Consistent with this premise, inhibitors of the anandamide-degrading enzyme fatty acid amide hydrolase (FAAH) produce analgesic and anxiolytic effects without cognitive defects. In contrast, we show that dual blockade of the endocannabinoid-degrading enzymes monoacylglycerol lipase (MAGL) and FAAH by selected organophosphorus agents leads to greater than ten-fold elevations in brain levels of both 2-AG and anandamide and to robust CB(1)-dependent behavioral effects that mirror those observed with CB(1) agonists. Arachidonic acid levels are decreased by the organophosphorus agents in amounts equivalent to elevations in 2-AG, which indicates that endocannabinoid and eicosanoid signaling pathways may be coordinately regulated in the brain.     

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO₂ uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.Energy security and climate change as global problems urge increased efforts to use plants as renewable energy sources both for power generation and transportation fuel production. Selected wood species, such as willows (Salix spp.), can be cultivated as short-rotation coppice for the rapid accumulation of biomass and reduction of CO₂ emission. Coppicing reinvigorates shoot growth, resulting in a special woody plant life cycle that differs from natural tree development, which takes decades. In this cultivation system, small stem cuttings are planted at high densities (15,000–25,000 ha⁻¹). In the soil, these dormant wood cuttings first produce roots and shoots that emerge from reactivated buds. During the first year, the growing shoots mature to woody stems. In the winter, these stems are cut back, and in the following spring, the cut stumps develop multiple shoots. The short-rotation coppice plantations are characterized by a very short, 2- to 3-year rotation, and the most productive varieties can produce up to 15 tons of oven-dried wood per hectare per year (Cunniff and Cerasuolo, 2011). The high-density willow plantations can also be efficiently used for heavy metal or organic phytoremediation, as reviewed by Marmiroli et al. (2011).The biomass productivity of shrub willows is largely dependent on coppicing capability, early vigorous growth, shoot growth rate and final stem height, root system size, photosynthetic efficiency, formation and composition of woody stems, water and nutrient use, as well as abiotic and biotic stress tolerance. Genetic improvement of all these traits can be based on broad natural genetic resources represented by more than 400 species in the genus Salix. More than 200 species have hybrid origins, and ploidy levels vary from diploid up to dodecaploid (Suda and Argus, 1968; Newsholme, 1992). In addition to molecular marker-assisted clone selection, intraspecific and interspecific crosses have been shown to further extend genetic variability in breeding programs for biomass yield (Karp et al., 2011).During natural diversification and artificial crossings of Salix spp., the willow genomes frequently undergo polyploidization, resulting in triploid or tetraploid allopolyploids. In triploid hybrids, both heterosis and ploidy can contribute to the improved biomass yield (Serapiglia et al., 2014). While the alloploid triploids have attracted considerable attention in willow improvement, the potentials of autotetraploid willow genotypes have not been exploited so far. As shown for other short-rotation wood species (poplar [Populus spp.], black locust [Robinia pseudoacacia], Paulownia spp., and birch [Betula spp.]), doubling the chromosome set by colchicine treatment can cause significant changes in organ morphology or growth parameters (Tang et al., 2010; Cai and Kang, 2011; Harbard et al., 2012; Mu et al., 2012; Wang et al., 2013a, 2013b). In several polyploidization protocols, the in vitro cultured tissues are exposed to different doses of colchicine or other inhibitors of mitotic microtubule function, and plantlets are differentiated from polyploid somatic cells (Tang et al., 2010; Cai and Kang, 2011). Alternatively, seeds or apical meristems of germinating seedlings can be treated with a colchicine solution (Harbard et al., 2012). Allotetraploids of poplar were produced by zygotic chromosome doubling that was induced by colchicine and high-temperature treatment (Wang et al., 2013a).Since tetraploid willow plants with 2n = 4x = 76 chromosomes are expected to represent novel genetic variability, especially for organ development and physiological parameters, a polyploidization project was initiated that was based on a highly productive diploid energy willow (S. viminalis var. Energo). Colchicine treatment of reactivated axillary buds of the in vitro-grown energy willow plantlets resulted in autotetraploid shoots and, subsequently, plants. For comparison of diploid and tetraploid variants of willow plants, digital imaging of green organs and roots was used for phenotyping. Among the tetraploid lines, genotypes were identified with improved biomass production, better photosynthetic parameters, and altered organ structure and hormone composition. The new tetraploid willow variants produced can serve as a unique experimental material to uncover key factors in biomass production in this short-rotation energy plant. In the future, these plants can also serve as crossing partners of diploid lines for the production of novel triploid energy willow genotypes.     

Inducible gene modification in the gastric epithelium of Tff1‐CreERT2, Tff2‐rtTA,Tff3‐luc mice

Temporal and spatial regulation of genes mediated by tissue‐specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium‐specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain confers tamoxifen‐inducible irreversible somatic recombination and allows simultaneous doxycycline‐dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1‐CreERT2 and Tff2‐rtTA transgene activity in a partially overlapping subset of long‐term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626–635, 2016. © 2016 Wiley Periodicals, Inc.     

Morphological variation of Keratella cochlearis (Gosse) in Lake Biwa,Japan

The length of the lorica (LL) of Keratella cochlearis cochlearis and of K. cochlearis tecta and the length of posterior spine (PSL) of the latter morphotype were measured in the strongly eutrophic basin and also in the mesotrophic basin of Lake Biwa, Japan, from September to December, 1980. In the population from the mesotrophic basin, the individuals with longer PSL prevail and the tecta forms are extremely rare. The LL values of both morphotypes from one sample do not differ. In December the LL increased to 95 µm in both morphotypes from 80 µm observed in September, while the PSL values decreased abrubtly in both basins in the middle of this period. It is suggested that the observed increase of LL could be related to the thermic factor, i.e. a steady decrease of water temperature, and the changes of PSL are correlated with the increase of nannoplankton and detritus aggregates noted in November. In this month an increase in fecundity and in the total numbers of rotifers took place as well (Hillbricht-Ilkowska, in press).