Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Morphological and Ultrastructural Features of Formation of the Skin of Wheat (Triticum aestivum L.) Kernel.

The integumentary tissues of plant seeds protect the embryo (new sporophyte) forming in them from unfavorable external conditions; therefore, comprehensive knowledge about the structural and functional specificity of seed covers in various plants may be of both theoretical and practical interest. As a result of our study, additional data were obtained on the morphological and ultrastructural features of the formation of a multilayer skin of wheat (Triticum aestivum L.) kernel (caryopsis). The ultrastructure research analysis showed that differentiation of the pericarp and inner integument of the ovule leads to the formation of functionally different layers of the skin of mature wheat grain. Thus, the differentiation of exocarp and endocarp cells is accompanied by a significant thickening of the cell walls, which reliably protect the ovule from adverse external conditions. The cells of the two-layer inner integument of the ovule differentiate into cuticular and phenolic layers, which are critical for protecting daughter tissues from various pathogens. The epidermis of the nucellus turns into a layer of mucilage, which apparently helps to maintain the water balance of the seed. Morphological and ultrastructural data showed that the formation of the kernel's skin occurs in coordination with the development of the embryo and endosperm up to the full maturity of the kernel. This is evidenced by the structure of the cytoplasm and nucleus, characteristic of metabolically active protoplasts of cells, which is observed in most integumentary layers at the late stages of maturation. This activity can also be confirmed by a significant increase in the thickness of the cell walls in the cells of two layers of the exocarp and in cross cells in comparison with the earlier stages. Based on these results, we came to the conclusion that the cells of a majority in the covering tissues of the wheat kernel during its ontogenesis are transformed into specialized layers of the skin by terminal differentiation.

Salt Stress-Induced Structural Changes Are Mitigated in Transgenic Tomato Plants Over-Expressing Superoxide Dismutase.

Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na2SO4 solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na2SO4 treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.

Development of a marmalade for patients with type 2 diabetes: Sensory characteristics and acceptability.

Type 2 diabetes is one of the most common noncommunicable diseases worldwide. The quality of life of people with this metabolic disorder is highly related to nutrition, given that products for glycemic control are of great importance for them. In this study, we have developed marmalades for glycemic control with the aims to investigate the most important sensory characteristics, to study the impact of the sensory properties on the acceptability of these marmalades, and to evaluate a difference in the acceptability of the marmalade samples between healthy people and people with type 2 diabetes. The main objects of the investigation were agar-, gelatin-, and pectin-based marmalades with maltitol, dried fruits, and berries for glycemic control. By means of descriptive sensory analysis, we have shown that major factors of the sensory differentiation of marmalade samples are the type of gelling agent and presence of nonsoluble components such as apple puree, which influencing the perception of "off-flavor," "gumminess," and "springiness" sensory attributes. Results of this research show that even with significant differences in sensory attributes it is possible to develop marmalade for glycemic control that will have no differences in the total liking score for the perception of both healthy people and patients with type 2 diabetes.

Consecutive entosis stages in human substrate-dependent cultured cells.

Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.

Polycationic ligands of different chemical classes stimulate DNA strand displacement between short oligonucleotides in a protein-free system.

The ability of polycationic ligands to stimulate DNA strand displacement between short oligonucleotides in a protein-free system is demonstrated. We show that two ligands, tetracationic aliphatic amine (spermine) and a dicationic intercalating drug (chloroquine), promote strand displacement in a concentration-dependent manner. At low concentrations both ligands decelerate spontaneous strand displacement because of their impact on the stability of the DNA duplex. At elevated concentrations they accelerate strand displacement via formation of intermediate structures containing three DNA strands. The rate of the last process does not correlate with the thermal dissociation rate of the entire DNA duplex. It indicates that, possibly, the action of these agents cannot be explained by their influence on the stability of the DNA duplex. In general, our results suggest that the ability to stimulate DNA strand displacement appears to be a common feature of polycations of different chemical and structural classes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 633-641, 2016.

Normobaric, intermittent hypoxia conditioning is cardio- and vasoprotective in rats.

Favorable versus detrimental cardiovascular responses to intermittent hypoxia conditioning (IHC) are heavily dependent on experimental or pathological conditions, including the duration, frequency and intensity of the hypoxia exposures. Recently, we demonstrated that a program of moderate, normobaric IHC (FIO2 9.5-10% for 5-10 min/cycle, with intervening 4 min normoxia, 5-8 cycles/day for 20 days) in dogs afforded robust cardioprotection against infarction and arrhythmias induced by coronary artery occlusion-reperfusion, but this protection has not been verified in other species. Accordingly, in this investigation cardio- as well as vasoprotection were examined in male Wistar rats completing the normobaric IHC program or a sham program in which the rats continuously breathed atmospheric air. Myocardial ischemia and reperfusion (IR) was imposed by occlusion and reperfusion of the left anterior descending coronary artery in in situ experiments and by subjecting isolated, perfused hearts to global ischemia-reperfusion. Cardiac arrhythmias and myocardial infarct size were quantified in in situ experiments. Endothelial function was evaluated from the relaxation to acetylcholine of norepinephrine-precontracted aortic rings taken from in situ IR experiments, and from the increase in coronary flow produced by acetylcholine in isolated hearts. IHC sharply reduced cardiac arrhythmias during ischemia and decreased infarct size by 43% following IR. Endothelial dysfunction in aorta was marked after IR in sham rats, but not significant in IHC rats. Similar findings were found for the coronary circulations of isolated hearts. These findings support the hypothesis that moderate, normobaric IHC is cardio- and vasoprotective in a rat model of IR.

Linker histone H1 stimulates DNA strand exchange between short oligonucleotides retaining high sensitivity to heterology.

The interaction of human linker histone H1(0) with short oligonucleotides was characterized. The capability of the histone to promote DNA strand exchange in this system has been demonstrated. The reaction is reversible at saturating amounts of H1 corresponding to complete binding of the oligonucleotide substrates with the histone. In our conditions the complete saturation of DNA with the histone occurs at a ratio of one protein molecule per about 60 nucleotides irrespectively of DNA strandedness. In contrast to the DNA strand exchange promoted by RecA-like enzymes of homologous recombination the H1 promoted reaction exhibits low tolerance to interruptions of homology between oligonucleotide substrates comparable to those for the case of spontaneous strand exchange between free DNA molecules at elevated temperatures and the exchange promoted by some synthetic polycations.

A high molecular weight polypeptide cross-reacting with the antibodies to the dynein heavy chain localizes to the subset of Golgi complex in higher plant cells.

Antibodies were produced against fragments of the microtubule-binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross-reacted with a polypeptide of high molecular weight (>500kDa). The antibodies bound to protein A-Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co-labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross-reacting with the antibodies to motor and microtubule-binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle-dependent manner. A subset of these aggregates belongs to the Golgi complex.

Reversibility, equilibration, and fidelity of strand exchange reaction between short oligonucleotides promoted by RecA protein from escherichia coli and human Rad51 and Dmc1 proteins.

We demonstrate the reversibility of RecA-promoted strand exchange reaction between short oligonucleotides in the presence of adenosine 5'-O-(thiotriphosphate). The reverse reaction proceeds without the dissociation of RecA from DNA. The reaction reaches equilibrium and its yield depends on the homology between the reaction substrates. We estimate the tolerance of the RecA-promoted strand exchange to individual base substitutions for a comprehensive set of possible base combinations in a selected position along oligonucleotide substrates for strand exchange and find, in agreement with previously reported estimations, that this tolerance is higher than in the case of free DNA. It is demonstrated that the short oligonucleotide-based approach can be applied to the human recombinases Rad51 and Dmc1 when strand exchange is performed in the presence of calcium ions and ATP. Remarkably, despite the commonly held belief that the eukaryotic recombinases have an inherently lower strand exchange activity, in our system their efficiencies in strand exchange are comparable with that of RecA. Under our experimental conditions, the human recombinases exhibit a significantly higher tolerance to interruptions of homology due to point base substitutions than RecA. Finding conditions where a chemical reaction is reversible and reaches equilibrium is critically important for its thermodynamically correct description. We believe that the experimental system described here will substantially facilitate further studies on different aspects of the mechanisms of homologous recombination.

Trifluralin-induced disorganization of microtubular cytoskeleton alters the development of roots in Hordeum vulgare L.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the toxicological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 microg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression--alteration that can be caused by cytoskeleton disorder. Using antibodies to alpha-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of microtubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.

Phasing of RecA monomers on quasi-random DNA sequences.

We show that some arbitrarily chosen DNA sequences have the ability to influence the positioning of RecA monomers in RecA-DNA complexes. The preferential phase of binding of RecA monomers is shown to depend on the DNA sequence and its nucleotide composition. A simple rearrangement of bases in a limited DNA stretch influences the phasing of RecA monomers. On the other hand, that some features of DNA sequences interfere with the phasing on specific DNA sites demonstrates the existence of mechanisms for both positive and negative regulation of phasing on natural DNAs. The possible role of phasing of RecA monomers on DNA is discussed.

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem.

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.