The Different Composition of Coumarins and Antibacterial Activity of Phlojodicarpus sibiricus and Phlojodicarpus villosus Root Extracts.

Phlojodicarpus sibiricus, a valuable endangered medicinal plant, is a source of angular pyranocoumarins used in pharmacology. Due to limited resource availability, other pyranocoumarin sources are needed. In the present research, the chemical composition of a closely related species, Phlojodicarpus villosus, was studied, along with P. sibiricus. High-performance liquid chromatography and mass-spectrometric analyses, followed by antibacterial activity studies of root extracts from both species, were performed. P. sibiricus and P. villosus differed significantly in coumarin composition. Pyranocoumarins predominated in P. sibiricus, while furanocoumarins predominated in P. villosus. Osthenol, the precursor of angular pyrano- and furanocoumarins, was detected in both P. sibiricus and P. villosus. Angular forms of coumarins were detected in both species according to the mass-spectrometric behavior of the reference. Thus, P. villosus cannot be an additional source of pyranocoumarins because their content in the plant is critically low. At the same time, the plant contained large amounts of hydroxycoumarins and furanocoumarins. The extracts exhibited moderate antibacterial activity against five standard strains. The P. villosus extract additionally suppressed the growth of the Gram-negative bacterium E. coli. Thus, both Phlojodicarpus species are promising for further investigation in the field of pharmaceuticals as producers of different coumarins.

Holothurian triterpene glycoside cucumarioside A2-2 induces macrophages activation and polarization in cancer immunotherapy.

BACKGROUND: Despite intensive developments of adoptive T cell and NK cell therapies, the efficacy against solid tumors remains elusive. Our study demonstrates that macrophage-based cell therapy could be a potent therapeutic option against solid tumors. METHODS: To this end, we determine the effect of a natural triterpene glycoside, cucumarioside A2-2 (CA2-2), on the polarization of mouse macrophages into the M1 phenotype, and explore the antitumor activity of the polarized macrophage. The polarization of CA2-2-pretreated macrophages was analyzed by flow cytometry and confocal imaging. The anti-cancer activity of CA2-2 macrophages was evaluated against 4T1 breast cancer cells and EAC cells in vitro and syngeneic mouse model in vivo. RESULTS: Incubation of murine macrophages with CA2-2 led to polarization into the M1 phenotype, and the CA2-2-pretreated macrophages could selectively target and kill various types of cancer in vitro. Notably, loading near-infrared (NIR) fluorochrome-labeled nanoparticles, MnMEIO-mPEG-CyTE777, into macrophages substantiated that M1 macrophages can target and penetrate tumor tissues in vivo efficiently. CONCLUSION: In this study, CA2-2-polarized M1 macrophages significantly attenuated tumor growth and prolonged mice survival in the syngeneic mouse models. Therefore, ex vivo CA2-2 activation of mouse macrophages can serve as a useful model for subsequent antitumor cellular immunotherapy developments.

Auxin-dependent regulation of growth via rolB-induced modulation of the ROS metabolism in the long-term cultivated pRiA4-transformed Rubiacordifolia L. calli.

Gene transfer from Agrobacterium to plants is the best studied example of horizontal gene transfer (HGT) between prokaryotes and eukaryotes. The rol genes of A. rhizogenes (Rhizobium rhizogenes) provide uncontrolled root growth, or "hairy root" syndrome, the main diagnostic feature. In the present study, we investigated the stable pRiA4-transformed callus culture of Rubia cordifolia L. While untransformed callus cultures need PGRs (plant growth regulators) as an obligatory supplement, pRiA4 calli is able to achieve long-term PGR-free cultivation. For the first time, we described the pRiA4-transformed callus cultures' PGR-dependent ROS status, growth, and specialized metabolism. As we have shown, expression of the rolA and rolB but not the rolC genes is contradictory in a PGR-dependent manner. Moreover, a PGR-free pRiA4 transformed cell line is characterised as more anthraquinone (AQ) productive than an untransformed cell culture. These findings pertain to actual plant biotechnology: it could be the solution to troubles in choosing the best PGR combination for the cultivation of some rare, medicinal, and woody plants; wild-type Ri-plants and tissue cultures may become freed from legal controls on genetically modified organisms in the future. We propose possible PGR-dependent relationships between rolA and rolB as well as ROS signalling targets. The present study highlighted the high importance of the rolA gene in the regulation of combined rol gene effects and the large knowledge gap in rolA action.

Enhanced Production of Nitrogenated Metabolites with Anticancer Potential in Aristolochia manshuriensis Hairy Root Cultures.

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism.

Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

Suppression of the HOS1 Gene Affects the Level of ROS Depending on Light and Cold.

The E3 ubiquitin-protein ligase HOS1 is an important integrator of temperature information and developmental processes. HOS1 is a negative regulator of plant cold tolerance, and silencing HOS1 leads to increased cold tolerance. In the present work, we studied ROS levels in hos1Cas9Arabidopsis thaliana plants, in which the HOS1 gene was silenced by disruption of the open reading frame via CRISPR/Cas9 technology. Confocal imaging of intracellular reactive oxygen species (ROS) showed that the hos1 mutation moderately increased levels of ROS under both low and high light (HL) conditions, but wild-type (WT) and hos1Cas9 plants exhibited similar ROS levels in the dark. Visualization of single cells did not reveal differences in the intracellular distribution of ROS between WT and hos1Cas9 plants. The hos1Cas9 plants contained a high basal level of ascorbic acid, maintained a normal balance between reduced and oxidized glutathione (GSH and GSSG), and generated a strong antioxidant defense response against paraquat under HL conditions. Under cold exposure, the hos1 mutation decreased the ROS level and substantially increased the expression of the ascorbate peroxidase genes Apx1 and Apx2. When plants were pre-exposed to cold and further exposed to HL, the expression of the NADPH oxidase genes RbohD and RbohF was increased in the hos1Cas9 plants but not in WT plants. hos1-mediated changes in the level of ROS are cold-dependent and cold-independent, which implies different levels of regulation. Our data indicate that HOS1 is required to maintain ROS homeostasis not only under cold conditions, but also under conditions of both low and high light intensity. It is likely that HOS1 prevents the overinduction of defense mechanisms to balance growth.

Plant Pyranocoumarins: Description, Biosynthesis, Application.

This overview article contains information about pyranocoumarins over the last 55 years. The article is based on the authors' phytochemical and physiological studies in vivo and in vitro as well as search and analysis of data in literature available on Google Scholar, Web of Science, PubMed, and ScienceDirect before January 2022. Pyranocoumarins are synthesized in plants of the Apiaceae, Rutaceae families, and one species in each of the Cornaceae, Calophyllaceae, and Fabaceae families can synthesize this class of compounds. The physiological role of these compounds in plants is not clear. It has been proven that these substances have a wide range of biological activities: anti-cancer, anti-spasmatic, and anticoagulant, and they also inhibit erythrocyte lysis and accumulation of triacylglycerides. The overview generalizes the modern understanding of the classification, structure, and biological activity of natural pyranocoumarins, and summarizes dispersed data into a unified scheme of biosynthesis. The review analyzes data on the localization and productivity of these substances in individual organs and the whole plant. It discusses a link between the unique structure of these substances and their biological activity, as well as new opportunities for pyranocoumarins in pharmacology. The article evaluates the potential of different plant species as producers of pyranocoumarins and considers the possibilities of cell cultures to obtain the end product.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

CRISPR/Cas9-Mediated Knockout of HOS1 Reveals Its Role in the Regulation of Secondary Metabolism in Arabidopsis thaliana.

In Arabidopsis, the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) functions as a main regulator of the cold signaling. In this study, CRISPR/Cas9-mediated targeted mutagenesis of the HOS1 gene in the first exon was performed. DNA sequencing showed that frameshift indels introduced by genome editing of HOS1 resulted in the appearance of premature stop codons, disrupting the open reading frame. Obtained hos1 Cas9 mutant plants were compared with the SALK T-DNA insertion mutant, line hos1-3, in terms of their tolerance to abiotic stresses, accumulation of secondary metabolites and expression levels of genes participating in these processes. Upon exposure to cold stress, enhanced tolerance and expression of cold-responsive genes were observed in both hos1-3 and hos1 Cas9 plants. The hos1 mutation caused changes in the synthesis of phytoalexins in transformed cells. The content of glucosinolates (GSLs) was down-regulated by 1.5-times, while flavonol glycosides were up-regulated by 1.2 to 4.2 times in transgenic plants. The transcript abundance of the corresponding MYB and bHLH transcription factors, which are responsible for the regulation of secondary metabolism in Arabidopsis, were also altered. Our data suggest a relationship between HOS1-regulated downstream signaling and phytoalexin biosynthesis.

Synthetic 1,4-Naphthoquinones inhibit P2X7 receptors in murine neuroblastoma cells.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel and potential therapeutic target for new drug development. In this study, we synthesized a series of new 1,4-naphthoquinone (1,4-NQ) derivatives and investigated their antagonistic effects against mouse P2X7R. We explored the ability of the tested substances to block ATP-induced Ca2+ influx into mouse Neuro-2a cells and selected the four most effective substances: the 1,4-naphthoquinone thioglucosides U-548 and U-557 and their tetracyclic conjugates U-286 and U-556. Biological analysis of these compounds revealed significant in vitro inhibition of murine P2X7R. This inhibition resulted in marked blockade of ethidium bromide (EtBr) and YO-PRO-1 fluorescent dye uptake, pronounced decreases in ROS and NO production and protection of neuronal cell viability against the toxic action of high ATP concentrations. In silico analysis indicated favorable molecular docking results of these 1,4-NQs, pointing to their potential to bind in an allosteric site located in the extracellular region of P2X7R. These findings suggest compounds U-286, U-548, U-556 and U-557 as potential scaffolds for the design of new P2X7R blockers and drugs effective against neuropathic pain and neurodegenerative diseases.

Age-Dependent and Tissue-Specific Alterations in the rDNA Clusters of the Panax ginseng C. A. Meyer Cultivated Cell Lines.

Long-term cultivation of Panax ginseng cell lines leads to a decreasing synthesis of the biologically active substances used in traditional medicine. To gain insight into the cellular mechanisms which may influence this process, we analyzed variations within the rDNA cluster of the Oriental ginseng cell lines. The cell lines were cultivated for 6 and 24 years; the number of nucleoli and chromosomes was analyzed. The complete 18S rDNA sequences were cloned and sequenced. The nucleotide polymorphism and phylogenetic relations of the sequences were analyzed, and the secondary structures for separate 18S rRNA regions were modeled. The 18S rDNA accumulated mutations during cell cultivation that correlate well with an increase in the number of chromosomes and nucleoli. The patterns of nucleotide diversity are culture-specific and the increasing polymorphism associates with cytosine methylation sites. The secondary structures of some 18S rRNA regions and their interaction can alter during cultivation. The phylogenetic tree topologies are particular for each cell line.The observed alterations in rDNA clusters are associated with a somaclonal variation, leading to changes in the pattern of intracellular synthesis during cell cultivation. The identified divergent rRNAs could provide additional gene expression regulation in P. ginseng cells by forming heterogeneous ribosomes.

Modulation of Doxorubicin Intracellular Accumulation and Anticancer Activity by Triterpene Glycoside Cucumarioside A2-2.

Keywords: cucumarioside A2-2; doxorubicin; ehrlich ascites carcinoma; MDR; sea cucumbers; triterpene glycosides.

Tempo-Spatial Pattern of Stepharine Accumulation in Stephania Glabra Morphogenic Tissues.

Alkaloids attract great attention due to their valuable therapeutic properties. Stepharine, an aporphine alkaloid of Stephania glabra plants, exhibits anti-aging, anti-hypertensive, and anti-viral effects. The distribution of aporphine alkaloids in cell cultures, as well as whole plants is unknown, which hampers the development of bioengineering strategies toward enhancing their production. The spatial distribution of stepharine in cell culture models, plantlets, and mature micropropagated plants was investigated at the cellular and organ levels. Stepharine biosynthesis was found to be highly spatially and temporally regulated during plant development. We proposed that self-intoxication is the most likely reason for the failure of the induction of alkaloid biosynthesis in cell cultures. During somatic embryo development, the toxic load of alkaloids inside the cells increased. Only specialized cell sites such as vascular tissues with companion cells (VT cells), laticifers, and parenchymal cells with inclusions (PI cells) can tolerate the accumulation of alkaloids, and thus circumvent this restriction. S. glabra plants have adapted to toxic pressure by forming an additional transport secretory (laticifer) system and depository PI cells. Postembryonic growth restricts specialized cell site formation during organ development. Future bioengineering strategies should include cultures enriched in the specific cells identified in this study.

Development of host strains and vector system for an efficient genetic transformation of filamentous fungi.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae.

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV-visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12-80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

CANINE DISTEMPER VIRUS IN A WILD FAR EASTERN LEOPARD ( PANTHERA PARDUS ORIENTALIS).

The critically endangered population of Far Eastern leopards ( Panthera pardus orientalis) may number as few as 60 individuals and is at risk from stochastic processes such as infectious disease. During May 2015, a case of canine distemper virus (CDV) was diagnosed in a wild leopard exhibiting severe neurologic disease in the Russian territory of Primorskii Krai. Amplified sequences of the CDV hemagglutinin gene and phosphoprotein gene aligned within the Arctic-like clade of CDV, which includes viruses from elsewhere in Russia, China, Europe, and North America. Histologic examination of cerebral tissue revealed perivascular lymphoid cuffing and demyelination of the white matter consistent with CDV infection. Neutralizing antibodies against CDV were detected in archived serum from two wild Far Eastern leopards sampled during 1993-94, confirming previous exposure in the population. This leopard population is likely too small to maintain circulation of CDV, suggesting that infections arise from spillover from more-abundant domestic or wild carnivore reservoirs. Increasing the population size and establishment of additional populations of leopards would be important steps toward securing the future of this subspecies and reducing the risk posed by future outbreaks of CDV or other infectious diseases.

Cucumarioside A2-2 Causes Macrophage Activation in Mouse Spleen.

The immunomodulatory effect of triterpene glycoside cucumarioside A2-2 (CA2-2), isolated from the Far Eastern sea cucumber Cucumaria japonica, was compared with lipopolysaccharide (LPS) on mouse spleen. It has been shown that the intraperitoneal (i.p.) glycoside administration leads to increased spleen macrophage activating markers iba-1, IL-1ß, iNOs, ROS and NO formation, with additional change of macrophage phenotype to M1. The mass spectrometry profiles of peptide/protein were obtained using MALDI-TOF-MS on the different parts of spleen sections isolated by laser mircodissection techniques. It was found that i.p. stimulation of animals with CA2-2 leads to marked changes in the intensity of the characteristic peaks of spleen peptides/proteins, primarily in red pulp.

Recent advances in the understanding of Agrobacterium rhizogenes-derived genes and their effects on stress resistance and plant metabolism.

It is commonly accepted that the plant pathogens Agrobacterium rhizogenes and Agrobacterium tumefaciens, acting via their T-DNA oncogenes, disturb hormone metabolism or hormone perception pathways in plants, thereby attaining their aim of successful pathogenesis. In this work, we summarize recent data on the A. rhizogenes rolC and rolB oncogenes in comparison to the A. tumefaciens 6b oncogene with respect to their effects on the physiology of transformed cells. The newly discovered functions of the rol genes include the modulation of secondary metabolism, the modulation of levels of intracellular ROS and stress resistance of transformed cells, changed sucrose metabolism, and the inhibition of programmed cell death. We show that the rol genes do not have suppressive effects on plant innate immunity; rather, these genes activate plant defense reactions. The existence of not only the hormone-related mechanism of pathogenicity but also the defense-related mechanism of pathogenicity during plant-Agrobacterium interactions is suggested.

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells.

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.