The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins.

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane-water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

L-DOPA ameliorates hippocampus-based mitochondria respiratory dysfunction caused by GCI/R injury.

Mitochondrial dysmorphology/dysfunction follow global cerebral ischemia-reperfusion (GCI/R) injury, leading to neuronal death. Our previous researches demonstrated that Levodopa (L-DOPA) improves learning and memory impairment in GCI/R rats by increasing synaptic plasticity of hippocampal neurons. This study investigates if L-DOPA, used in Parkinson's disease treatment, alleviates GCI/R-induced cell death by enhancing mitochondrial quality. Metabolomics and transcriptomic results showed that GCI/R damage affected the Tricarboxylic acid (TCA) cycle in the hippocampus. The results of this study show that L-DOPA stabilized mitochondrial membrane potential and ultrastructure in hippocampus of GCI/R rats, increased dopamine level in hippocampus, decreased succinic acid level, and stabilized Ca2+ level in CA1 subregion of hippocampus. As a precursor of dopamine, L-DOPA is presumed to improves mitochondrial function in hippocampus of GCI/R rats. However, dopamine cannot cross the blood-brain barrier, so L-DOPA is used in clinical therapy to supplement dopamine. In this investigation, OGD/R models were established in isolated mouse hippocampal neurons (HT22) and primary rat hippocampal neurons. Notably, dopamine exhibited a multifaceted impact, demonstrating inhibition of mitochondrial reactive oxygen species (mitoROS) production, stabilization of mitochondrial membrane potential and Ca2+ level, facilitation of TCA circulation, promotion of aerobic respiratory metabolism, and downregulation of succinic acid-related gene expression. Consistency between in vitro and in vivo results underscores dopamine's significant neuroprotective role in mitigating mitochondrial dysfunction following global cerebral hypoxia and ischemia injury. Supplement dopamine may represent a promising therapy to the cognitive impairment caused by GCI/R injury.

Epoxyalcohol Synthase Branch of Lipoxygenase Cascade.

Oxylipins are one of the most important classes of bioregulators, biosynthesized through the oxidative metabolism of unsaturated fatty acids in various aerobic organisms. Oxylipins are bioregulators that maintain homeostasis at the cellular and organismal levels. The most important oxylipins are mammalian eicosanoids and plant octadecanoids. In plants, the main source of oxylipins is the lipoxygenase cascade, the key enzymes of which are nonclassical cytochromes P450 of the CYP74 family, namely allene oxide synthases (AOSs), hydroperoxide lyases (HPLs), and divinyl ether synthases (DESs). The most well-studied plant oxylipins are jasmonates (AOS products) and traumatin and green leaf volatiles (HPL products), whereas other oxylipins remain outside of the focus of researchers' attention. Among them, there is a large group of epoxy hydroxy fatty acids (epoxyalcohols), whose biosynthesis has remained unclear for a long time. In 2008, the first epoxyalcohol synthase of lancelet Branchiostoma floridae, BfEAS (CYP440A1), was discovered. The present review collects data on EASs discovered after BfEAS and enzymes exhibiting EAS activity along with other catalytic activities. This review also presents the results of a study on the evolutionary processes possibly occurring within the P450 superfamily as a whole.

Curcumin as a Potential Antioxidant in Stress Regulation of Terrestrial, Avian, and Aquatic Animals: A Review.

Stress has brought about a variety of harmful impacts on different animals, leading to difficulties in the management of animal husbandry and aquaculture. Curcumin has been recognized as a potential component to ameliorate the adverse influence of animal stress induced by toxicity, inflammation, diseases, thermal effect, and so on. In detail, this compound is known to offer various outstanding functions, including antibacterial properties, antioxidant effects, immune response recovery, and behavioral restoration of animals under stress conditions. However, curcumin still has some limitations, owing to its low bioavailability. This review summarizes the latest updates on the regulatory effects of curcumin in terms of stress management in terrestrial, avian, and aquatic animals.

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.

Recombinant Soybean Lipoxygenase 2 (GmLOX2) Acts Primarily as a ω6(S)-Lipoxygenase.

The lipoxygenase (LOX) cascade is a source of bioactive oxylipins that play a regulatory role in plants, animals, and fungi. Soybean (Glycine max (L.) Merr.) LOXs are the classical models for LOX research. Progress in genomics has uncovered a large diversity of GmLOX isoenzymes. Most of them await biochemical investigations. The catalytic properties of recombinant soybean LOX2 (GmLOX2) are described in the present work. The GmLOX2 gene has been cloned before, but only for nucleotide sequencing, while the recombinant protein was not prepared and studied. In the present work, the recombinant GmLOX2 behavior towards linoleic, α-linolenic, eicosatetraenoic (20:4), eicosapentaenoic (20:5), and hexadecatrienoic (16:3) acids was examined. Linoleic acid was a preferred substrate. Oxidation of linoleic acid afforded 94% optically pure (13S)-hydroperoxide and 6% racemic 9-hydroperoxide. GmLOX2 was less active on other substrates but possessed an even higher degree of regio- and stereospecificity. For example, it converted α-linolenic acid into (13S)-hydroperoxide at about 98% yield. GmLOX2 showed similar specificity towards other substrates, producing (15S)-hydroperoxides (with 20:4 and 20:5) or (11S)-hydroperoxide (with 16:3). Thus, the obtained data demonstrate that soybean GmLOX2 is a specific (13S)-LOX. Overall, the catalytic properties of GmLOX2 are quite similar to those of GmLOX1, but pH is optimum.

Shellfish sanitation monitoring in La Spezia gulf: Chemometric evaluation of data from 2015 to 2021.

Shellfish sanitary controls are very important to guarantee consumer health because bivalve molluscs (BVM) are filter-feeders so they can accumulate pathogens, environmental contaminants and biotoxins produced by some algae, causing infections and food poisoning in humans after ingestion. The purpose of this work was to analyse with chemometric methods the historical data relating to routine analyses carried out by the competent authority (Liguria Local Health Unit, National Health Service) on the BVM reared in a shellfish farm located in the Gulf of La Spezia (Italy). Chemometric analysis was aimed at identifying any correlations between the variables, as well as any seasonal trends and similarities between the stations, in order to be able to provide further material for a more accurate risk assessment and to improve the monitoring organization for example by reducing sampling stations and/or sampling frequency. The dataset used included 31 variables classified as biotoxicological, microbiological and chemical variables, measured twice a week, monthly or half yearly respectively, for a total of 6 years (from 2015 to 2021), on samples of Mytilus galloprovincialis coming from 7 monitoring stations. The results obtained by the application of principal component analysis have shown positive alga-biotoxin correlations, as well as seasonal trends linked to algae growth, with a greater algal biomass and their toxins during the spring months. In addition, periods characterised by low rainfall were found to affect algal development, promoting especially species such as Dinophysis spp. Considering the microbiological and biotoxicological variables, significant differences between the monitoring stations were not found. However, stations could be distinguished on the basis of the nature of the predominant chemical pollutants.

Effect of Cytochrome C on the Conductance of Asolectin Membranes and the Occurrence of Through Pores at Different pHs.

The study of the electrical parameters of asolectin bilayer lipid membranes in the presence of cytochrome c (cyt c) at various concentrations showed that an increase in the concentration of cyt c leads to an increase in the membrane conductance and the appearance of through pores. The studied membranes did not contain cardiolipin, which is commonly used in studying the effect of cyt c on membrane permeability. In the presence of cyt c, discrete current fluctuations were recorded. The occurrence of these fluctuations may be associated with the formation of through pores. The diameter of these pores was ~0.8 nm, which is smaller than the size of the cyt c globule (~3 nm). Measurements carried out at pH values from 6.4 to 8.4 showed that the concentration dependence of the membrane conductance increases with increasing pH. To assess the binding of cyt c to the bilayer, we measured the concentration and pH dependences of the difference in surface potentials induced by the unilateral addition of cyt c. The amount of bound cyt c at the same concentrations decreased with increasing pH, which did not correspond to the conductance trend. An analysis of conductance traces leads to the conclusion that an increase in the integral conductance of membranes is associated with an increase in the lifetime of pores. The formation of "long-lived" pores, of which the residence time in the open state is longer than in the closed state, was achieved at various combinations of pHs and cyt c concentrations: the higher the pH, the lower the concentration at which the long-lived pores appeared and, accordingly, a higher conductance was observed. The increase in conductance and the formation of transmembrane pores are not due to the electrostatic interaction between cyt c and the membrane. We hypothesize that an increase in pH leads to a weakening of hydrogen bonds between lipid heads, which allows cyt c molecules to penetrate into the membrane. This disrupts the order of the bilayer and leads to the occurrence of through pores.

The impact of curcumin on livestock and poultry animal's performance and management of insect pests.

Plant-based natural products are alternative to antibiotics that can be employed as growth promoters in livestock and poultry production and attractive alternatives to synthetic chemical insecticides for insect pest management. Curcumin is a natural polyphenol compound from the rhizomes of turmeric (Curcuma spp.) and has been suggested to have a number of therapeutic benefits in the treatment of human diseases. It is also credited for its nutritional and pesticide properties improving livestock and poultry production performances and controlling insect pests. Recent studies reported that curcumin is an excellent feed additive contributing to poultry and livestock animal growth and disease resistance. Also, they detailed the curcumin's growth-inhibiting and insecticidal activity for reducing agricultural insect pests and insect vector-borne human diseases. This review aims to highlight the role of curcumin in increasing the growth and development of poultry and livestock animals and in controlling insect pests. We also discuss the challenges and knowledge gaps concerning curcumin use and commercialization as a feed additive and insect repellent.

A Review of the Role of Curcumin in Metal Induced Toxicity.

Metal toxicity poses a potential global threat to the environment and living beings. Their numerous agricultural, medical, industrial, domestic, and technological applications result in widespread distribution in the environment which raises concern on the potential effects of metals in terms of health hazards and environmental pollution. Chelation therapy has been the preferred medical treatment for metal poisoning. The chelating agent bounds metal ions to form complex cyclic structures known as 'chelates' to intensify their excretion from the body. The main disadvantage of synthetic chelators is that the chelation process removes vital nutrients along with toxic metals. Natural compounds are widely available, economical, and have minimal adverse effects compared to classical chelators. Herbal preparations can bind to the metal, reduce its absorption in the intestines, and facilitate excretion from the body. Curcumin, a bioactive substance in turmeric, is widely used as a dietary supplement. Most studies have shown that curcumin protects against metal-induced lipid peroxidation and mitigates adverse effects on the antioxidant system. This review article provides an analysis to show that curcumin imparts promising metal toxicity-ameliorative effects that are related to its intrinsic antioxidant activity.

Bilayer Lipid Membrane as Memcapacitance: Capacitance-Voltage Pinched Hysteresis and Negative Insertion Conductance.

Inelastic (dissipative) effects of different natures in lipid bilayer membranes can lead to hysteresis phenomena. Early, it was shown that lipid bilayer membranes, under the action of a periodic sinusoidal voltage, demonstrate pinched-hysteresis loops in the experimental capacitance-voltage dependences and are almost the only example of the physical implementation of memcapacitance. Here, we propose an equivalent circuit and mathematical framework for analyzing the dynamic nonlinear current response of a lipid bilayer membrane as an externally controlled memcapacitance. Solving a nonlinear differential equation for the equivalent circuit of a membrane in the form of a parallel connection of a nonlinear viscoelastic capacitor and an active resistance using the small parameter method, we obtain explicit analytical dependences for the current response of the membrane and pinched-hysteresis loops. The explicit solutions and their comparison with experimental data allow us to identify the lumped equivalent circuit parameters that govern the memcapacitor behavior of the membrane and hence the magnitude of the hysteresis. We quantify the memcapacitance hysteresis in terms of negative work done by the control signal. An analysis of the formulas leads to the conclusion that the determining factor for the appearance of pinched hysteresis is the type of nonlinear dependence of the device capacitance on voltage.

Effect of Cobalt Ferrite Nanoparticles in a Hydrophilic Shell on the Conductance of Bilayer Lipid Membrane.

We measured the conductance of bilayer lipid membranes of diphytanoylphosphatidylcholine induced by interaction with cubic magnetic nanoparticles (MNPs) of cobalt ferrite 12 and 27 nm in size and coated with a hydrophilic shell. The MNP coating is human serum albumin (HSA) or polyethylene glycol (PEG). The interaction of nanoparticles added to the bulk solution with the lipid bilayer causes the formation of metastable conductive pores, which, in turn, increases the integral conductance of the membranes. The increase in conductance with increasing MNP concentration was practically independent of the particle size. The dependence of the bilayer conductance on the concentration of PEG-coated MNPs was much weaker than that on the concentration with a shell of HSA. Analyzing the current traces, we believe that the conductive pores formed as a result of the interaction of nanoparticles with the membrane can change their size, remaining metastable. The form of multilevel current traces allows us to assume that there are several metastable pore states close in energy. The average radius of the putative cylindrical pores is in the range of 0.4-1.3 nm.

The Role of Endoplasmic Reticulum Stress in Differentiation of Cells of Mesenchymal Origin.

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states.

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins 'ectocarpins' by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus.

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.

Mechanism of Energy Storage and Transformation in the Mitochondria at the Water-Membrane Interface.

In this review, we discuss the mechanisms of generation of membrane-bound protons using different energy sources in model and natural systems. Analysis of these mechanisms revealed that all three types of reactions include the same principal stage, which is dissociation of electrically neutral Brønsted acids at the interface during transition from the hydrophobic phase to water with a low dielectric constant. Special attention is paid to the fact that in one of the analyzed model systems, membrane-bound protons provide energy for the reaction of ATP synthesis. Similar mechanism for the generation of membrane-bound protons has been found in natural membranes involved in oxidative phosphorylation, in particular, on the membranes of mitoplasts and mitochondria. The energy of oxidative reactions required for ATP synthesis, is stored at the intermediate stage not only in the form of transmembrane electrochemical potential of protons, but also and perhaps mostly, as protons attached to the inner mitochondrial membrane. The process of energy storage in mitochondria is linked to the transfer of protons that simultaneously perform two functions. Protons on the membrane surface carry free energy and, at the same time, act as substrates facilitating the movement of F1F0-ATP-synthase biological machine.

Ictal wavefront propagation in slices and simulations with conductance-based refractory density model.

The mechanisms determining ictal discharge (ID) propagation are still not clear. In the present study, we aimed to examine these mechanisms in animal and mathematical models of epileptiform activity. Using double-patch and extracellular potassium ion concentration recordings in rat hippocampal-cortical slices, we observed that IDs moved at a speed of about 1 mm/s or less. The mechanisms of such slow propagation have been studied with a mathematical, conductance-based refractory density (CBRD) model that describes the GABA- and glutamatergic neuronal populations' interactions and ion dynamics in brain tissue. The modeling study reveals two main factors triggerring IDs: (i) increased interneuronal activity leading to chloride ion accumulation and a consequent depolarizing GABAergic effect and (ii) the elevation of extracellular potassium ion concentration. The local synaptic transmission followed by local potassium ion extrusion and GABA receptor-mediated chloride ion accumulation underlies the ID wavefront's propagation. In contrast, potassium ion diffusion in the extracellular space is slower and does not affect ID's speed. The short discharges, constituting the ID, propagate much faster than the ID front. The accumulation of sodium ions inside neurons due to their hyperactivity and glutamatergic currents boosts the Na+/K+ pump, which terminates the ID. Knowledge of the mechanism of ID generation and propagation contributes to the development of new treatments against epilepsy.

Differential modulation of the lipoxygenase cascade during typical and latent Pectobacterium atrosepticum infections.

BACKGROUND AND AIMS: Plant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant-P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria. METHODS: Tobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches. KEY RESULTS: The obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity. CONCLUSIONS: Our results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).

Gibberellic Acid Initiates ER Stress and Activation of Differentiation in Cultured Human Immortalized Keratinocytes HaCaT and Epidermoid Carcinoma Cells A431.

Diterpenoid plant hormone gibberellic acid (GA) plays an important role in regulation of plant growth and development and is commonly used in agriculture for activation of plant growth and food production. It is known that many plant-derived compounds have miscellaneous biological effects on animals and humans, influencing specific cellular functions and metabolic pathways. However, the effect of GA on animal and human cells remains controversial. We investigated the effect of GA on cultured human cell lines of epidermoid origin-immortalized non-tumorigenic keratinocytes HaCaT and carcinoma A431 cells. We found that at a non-toxic dose, GA upregulated the expression of genes associated with the ER stress response-CHOP, sXBP1, GRP87 in both cell lines, and ATF4 predominantly in A431 cells. We also showed that GA was more effective in upregulating the production of ER stress marker GRP78, autophagy marker LC3B-II, and differentiation markers involucrin and filaggrin in A431 cells than in HaCaT. We conclude that GA induces mild ER stress in both cell lines, followed by the activation of differentiation via upregulation of autophagy. However, in comparison with immortalized keratinocytes HaCaT, GA is more effective in inducing differentiation of carcinoma A431 cells, probably due to the inherently lower differentiation status of A431 cells. The activation of differentiation in poorly differentiated and highly malignant A431 cells by GA may lower the level of malignancy of these cells and decrease their tumorigenic potential.

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.

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata).

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.