Volume 20, Issue 1

Stress tolerance in plants is a coordinated action of multiple stress response genes that also cross talk with other components of the stress signal transduction pathways. The expression and regulation of stress-induced genes are largely regulated by specific transcription factors, families of which have been reported in several plant species, such as Arabidopsis, rice and Populus. In sorghum, the majority of such factors remain unexplored. We used 2DE refined with MALDI-TOF techniques to analyze drought stress-induced proteins in sorghum. A total of 176 transcription factors from the MYB, AUX_ARF, bZIP, AP2 and WRKY families of drought-induced proteins were identified. We developed a method based on semantic similarity of gene ontology terms (GO terms) to identify the transcription factors. A threshold value (≥ 90%) was applied to retrieve total 1,493 transcription factors with high semantic similarity from selected plant species. It could be concluded that the identified transcription factors regulate their target proteins with endogenous signals and environmental cues, such as light, temperature and drought stress. The regulatory network and cis-acting elements of the identified transcription factors in distinct families are involved in responsiveness to auxin, abscisic acid, defense, stress and light. These responses may be highly important in the modulation of plant growth and development.

The antioxidant activities of five alkylresorcinol (AR) homologs with alkyl chains of 1, 3, 5 6 and 12 carbon atoms were studied using molecular and cellular assays for superoxide anions (O 2 .- ). The effect of ARs as superoxide anion scavengers was assessed using the photochemical reaction of spontaneous photo-reduced flavin re-oxidation. In this system, ARs reaction with O 2 .- produced dye derivatives, as C 6 - and C 12 -AR prevented the O 2 .- -induced conversion of nitroblue tetrazolium into formazan in AR-containing mixtures. The influence of ARs on soxS gene expression and bacterial cell viability was studied with the luminescent Escherichia coli K12 MG1655 psoxS’::luxCDABE-Amp R strain, showing low basal light emission. This increased significantly during paraquatinduced oxidative stress as a consequence of the simultaneous transcription of soxS-gene and lux-gene fusion. ARs with alkyl chains containing 5-12 carbon atoms at concentrations of 0.1-1.0 μM weakly induced soxS-gene expression, whereas 1-10 mM repressed it. This respectively increased or decreased the bacterial cell resistance to O 2 .- -related oxidative stress. AR derivatives lost their protective activity from reactions with superoxide anions, which required increased soxS gene expression for cell viability. These results show the dual nature of ARs, which possess direct antioxidant properties and the ability to indirectly regulate the activity of cellular antioxidative defense mechanisms.

Lysophosphatidic acid (LPA) and its analogs are well-known mitogens for various cell types. Many reports have confirmed that several types of cancer cell produce LPA to promote survival, growth and tumorigenesis. This indicates that the interface between the LPA signaling pathway and the cell cycle signaling system is critical to the control of cancer cell proliferation. However, our previous study indicated that cyclic phosphatidic acid (cPA), which is structurally similar to LPA, inhibits the proliferation and migration of colon cancer cells. It has been reported that cPA shows several biological activities not shown by LPA. However, understanding of the detailed molecular and cellular mechanism underlying the regulation of the cell cycle by cPA is still in its infancy. In this study, we investigated the effect of cPA treatment on human DLD-1 colon cancer cells by analyzing cell cycle dynamics, gene expression, and AKT phosphorylation. Our findings indicate that cPA inhibits cell cycle progression in DLD-1 colon cancer cells via the downregulation of cyclin D1 and the inhibition of AKT phosphorylation.

Human APEX nuclease 1 (APEX1) plays an important role in the repair of oxidative DNA lesions through base excision repair. It may influence the development of oxidative stress-related diseases. The aim of this study was to determine the relationship between the genotypes of the c.444 T>G (rs1130409) and c.-468 T>G (rs1760944) polymorphisms in the APEX1 gene and the occurrence of two oxidative stress-related eye diseases: keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). The study involved 250 patients with KC, 209 patients with FECD, and 350 control subjects. All of the patients and control subjects underwent a detailed ophthalmic examination. The polymorphisms were genotyped by mismatch polymerase chain reaction restriction fragment length polymorphism (mismatch PCR-RFLP). We observed that the G/T and T/T genotypes of the c.-468 T>G polymorphism were respectively associated with a decreased occurrence of KC (OR 0.54, 95% CI 0.37-0.95; p = 0.030) and an increased occurrence of KC (OR 1.87, 95% CI 1.06-3.32; p = 0.032). None of these polymorphisms showed any association with FECD. Furthermore, no other association was observed, including haplotypes of the two polymorphisms. Our findings suggest that the c.-468 T>G polymorphism of the APEX1 gene may play a role in the pathogenesis of KC.

FE65 is a brain-enriched, developmentally regulated adaptor protein that was first identified as a binding partner of amyloid precursor protein (APP), an important molecule in Alzheimer’s disease. FE65 possesses three protein interaction domains, including an N-terminal WW domain and two C-terminal phosphotyrosine-binding (PTB) domains. It is capable of mediating the assembly of multimolecular complexes. Although initial work reveals its roles in APP processing and gene transactivation, increasing evidence suggests that FE65 participates in more diverse biological processes than originally anticipated. This article discusses the role of FE65 in signal transduction during cell stress and protein turnover through the ubiquitin-proteasome system and in various neuronal processes, including neurogenesis, neuronal migration and positioning, neurite outgrowth, synapse formation and synaptic plasticity, learning, and memory.

STAT3 (signal transducer and activator of transcription 3) is an important cellular effector in the Jak/STAT signaling pathway, which plays a pivotal role in human immune system regulation, mediating the effect of different cytokines. In the present study, we assessed the correlation between STAT3 polymorphisms (rs3816769 C>T and rs744166 A>G) and risk of the autoimmune thyroid diseases (AITDs) Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) in the Polish population. Moreover, we evaluated the association of polymorphisms with the thyroid autoantibody levels (TPOAb, TgAb, TRAb) and the correlation between circulating proinflammatory IL6 and IL17 cytokines and thyroid autoantibody levels. The study included 71 AITD patients with HT (n = 39) or GD (n = 32) and a control group (n = 40). DNA SNP genotyping was performed using TaqMan probes. Serum levels of thyroid autoantibodies, IL6 and IL17 were measured according to enhanced chemiluminescence (ECL) assay. Allele A of STAT3 SNP rs744166 A>G was significantly more frequent in both HT and GD patients, while allele G was significantly more frequent in the control group. Similarly, allele C and CC genotype of STAT3 SNP rs3816769 C>T were significantly more frequent in the control group in comparison to HT and GD patients. Significantly higher TgAb median values were associated with CT rs3816769 genotype in HT patients. Serum levels of IL6 and IL17 positively correlated with TPOAb in the HT group. Serum level of IL6 positively correlated with TPOAb in the AITD group. Both studied polymorphisms seem to play a significant role in susceptibility to AITD (HT and GD). STAT3 SNPs may influence TAb level in AITD patients.

The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H 2 O 2 -induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H 2 O 2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H 2 O 2 -induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H 2 O 2 .

Extracellular vesicles (EVs), including exosomes, are a subject of intense interest due to their emission by cancer cells and role in intercellular communication. Earlier reports suggested that oncogenes, such as RAS, MET or EGFR, drive cellular vesiculation. Interestingly, these oncogenes may also traffic between cells using the EV-mediated emission and uptake processes. One of the main tools in the analysis of EVs are ultracentrifugation protocols designed to efficiently separate parental cells from vesicles through a sequence of steps involving increasing g-force. Here we report that ultracentrifugationonly EV preparations from highly transformed cancer cells, driven by the overexpression of oncogenic H-ras (RAS-3) and v-src (SRC-3), may contain clonogenic cancer cells, while preparations of normal or less aggressive human cell lines are generally free from such contamination. Introduction of a filtration step eliminates clonogenic cells from the ultracentrifugate. The survival of RAS-3 and SRC-3 cells under extreme conditions of centrifugal force (110,000 g) is oncogene-induced, as EV preparations of their parental non-tumourigenic cell line (IEC-18) contain negligible numbers of clonogenic cells. Moreover, treatment of SRC-3 cells with the SRC inhibitor (PP2) markedly reduces the presence of such cells in the unfiltered ultracentrifugate. These observations enforce the notion that EV preparations require careful filtration steps, especially in the case of material produced by highly transformed cancer cell types. We also suggest that oncogenic transformation may render cells unexpectedly resistant to extreme physical forces, which may affect their biological properties in vivo.

Pulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn’s disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers.The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients. The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis.The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation. A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL.The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank’s solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

The promoter of skeletal muscle α-actin gene (ACTA1) is highly muscle specific. The core of the bovine ACTA1 promoter extends from +29 to −233, about 262 base pairs (bp), which is sufficient to activate transcription in bovine muscle satellite cells. In this study, analysis by PCR site-specific mutagenesis showed that the cis-acting element SRE (serum response element binding factor) was processed as a transcriptional activator. In order to enhance the bovine ACTA1 promoter’s activity, we used a strategy to modify it. We cloned a fragment containing three SREs from the promoter of ACTA1, and then one or two clones were linked upstream of the core promoter (262 bp) of ACTA1. One and two clones increased the activity of the ACTA1 promoter 3-fold and 10-fold, respectively, and maintained muscle tissue specificity. The modified promoter with two clones could increase the level of ACTA1 mRNA and protein 4-fold and 1.1-fold, respectively. Immunofluorescence results showed that green fluorescence of ACTA1 increased. Additionally, the number of total muscle microfilaments increased. These genetically engineered promoters might be useful for regulating gene expression in muscle cells and improving muscle mass in livestock.