Improvement of lidocaine skin permeation by using passive and active enhancer methods.

Lidocaine is generally recognized and preferred for local anaesthesia, but in addition, studies have described additional benefits of lidocaine in cancer therapy, inflammation reduction, and wound healing. These properties contribute to its increasing importance in dermatological applications, and not only in pain relief but also in other potential therapeutic outcomes. Therefore, the purpose of our study was to enhance lidocaine delivery through the skin. A stable nanostructured lipid carrier (NLC), as a passive permeation enhancer, was developed using a 23 full factorial design. The nanosystems were characterized by crystallinity behaviour, particle size, zeta potential, encapsulation efficiency measurements, and one of them was selected for further investigation. Then, NLC gel was formulated for dermal application and compared to a traditional dermal ointment in terms of physicochemical (rheological behaviour) and biopharmaceutical (qualitative Franz diffusion and quantitative Raman investigations) properties. The study also examined the use of 3D printed solid microneedles as active permeation enhancers for these systems, offering a minimally invasive approach to enhance transdermal drug delivery. By actively facilitating drug permeation through the skin, microneedles can complement the passive transport achieved by NLCs, thereby providing an innovative and synergistic approach to improving lidocaine delivery.

Prognostic potential of CUL3 ligase with differential roles in luminal A and basal type breast cancer tumors.

Breast cancer is a prevalent and significant cause of mortality in women, and manifests as six molecular subtypes. Its further histologic classification into non-invasive ductal or lobular carcinoma (DCIS) and invasive carcinoma (ILC or IDC) underscores its heterogeneity. The ubiquitin-proteasome system plays a crucial role in breast cancer, with inhibitors targeting the 26S proteasome showing promise in clinical treatment. The Cullin-RING ubiquitin ligases, including CUL3, have direct links to breast cancer. This study focuses on CUL3 as a potential biomarker, leveraging high-throughput sequencing, gene expression profiling, experimental and data analysis tools. Through comprehensive analysis using databases like GEPIA2 and UALCAN, as well as TCGA datasets, CUL3's expression and its association with prognostic values were assessed. Additionally, the impact of CUL3 overexpression was explored in MCF-7 and MDA-MB-231 breast cancer cell lines, revealing distinct differences in molecular and phenotypic characteristics. We further profiled its expression and localization in breast cancer tissues identifying prominent differences between luminal A and TNBC tumors. Conclusively, CUL3 was found to be associated with cell cycle progression, and DNA damage response, exhibiting diverse roles depending on the tumor's molecular type. It exhibits a tendency to act as an oncogene in triple-negative tumors and as a tumor suppressor in luminal A types, suggesting a potential significance in breast cancer progression and therapeutic directions.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants.

Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63-19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division-elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin-cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes.

General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA- and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes.

Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

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.

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas.

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1.

The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

Rac1 participates in thermally induced alterations of the cytoskeleton, cell morphology and lipid rafts, and regulates the expression of heat shock proteins in B16F10 melanoma cells.

Eukaryotic cells exhibit a characteristic response to hyperthermic treatment, involving morphological and cytoskeletal alterations and the induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. We addressed here how small GTPase Rac1 integrates signals from heat stress and simultaneously induces various cellular changes in mammalian cells. As evidence that Rac1 is implicated in the heat shock response, we first demonstrated that both mild (41.5°C) and severe (43°C) heat shock induced membrane translocation of Rac1. Following inhibition of the activation or palmitoylation of Rac1, the size of its plasma membrane-bound pool was significantly decreased while the heat shock-induced alterations in the cytoskeleton and cell morphology were prevented. We earlier documented that the size distribution pattern of cholesterol-rich rafts is temperature dependent and hypothesized that this is coupled to the triggering mechanism of stress sensing and signaling. Interestingly, when plasma membrane localization of Rac1 was inhibited, a different and temperature independent average domain size was detected. In addition, inhibition of the activation or palmitoylation of Rac1 resulted in a strongly decreased expression of the genes of major heat shock proteins hsp25 and hsp70 under both mild and severe heat stress conditions.

Assessing the Applicability of Singlet Oxygen Photosensitizers in Leaf Studies.

Singlet oxygen ((1) O2 ) is of special interest in plant stress physiology. Studies focused on internal, chlorophyll-mediated production are often complemented with the use of artificial (1) O2 photosensitizers. Here, we report a comparative study on the effects of Rose Bengal (RB), Methylene Violet (MVI), Neutral Red (NR) and Indigo Carmine (IC). These were infiltrated into tobacco leaves at concentrations generating the same fluxes of (1) O2 in solution. Following green light-induced (1) O2 production from these dyes, leaf photosynthesis was characterized by Photosystem (PS) II and PSI electron transport and oxidative damage was monitored as degradation of D1, a PSII core protein. Cellular localizations were identified on the basis of the dyes' fluorescence using confocal laser scanning microscopy. We found that RB and NR were both localized in chloroplasts but the latter had very little effect, probably due to its pH-dependent photosensitizing. Both RB and intracellular, nonplastid MVI decreased PSII electron transport, but the effect of RB was stronger than that of MVI and only RB was capable of damaging the D1 protein. Intercellularly localized IC had no significant effect. Our results also suggest caution when using RB as photosensitizer because it affects PSII electron transport.

Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. RESULTS: In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. CONCLUSION: These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids.

Synthesis and potential use of 1,8-naphthalimide type (1)O2 sensor molecules.

New double (fluorescent and spin) sensor molecules containing 4-amino substituted 1,8-naphthalimide as a fluorophore and a sterically hindered amine (pre-nitroxide) or pyrroline nitroxide as a quencher and radical capturing moiety were synthesized. All sensors were substituted with a diethylaminoethyl side-chain to increase the water solubility. Steady state fluorescence properties of these compounds and their responses to ROS in vitro are reported with perspectives of plant physiology use in vivo.

Identification of transcriptional and phosphatase regulators as interaction partners of human ADA3, a component of histone acetyltransferase complexes.

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase II-mediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners.

Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts.

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.