Evaluation of the significance of cell wall polymers in flax infected with a pathogenic strain of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum infection leads to Fusarium-derived wilt, which is responsible for the greatest losses in flax (Linum usitatissimum) crop yield. Plants infected by Fusarium oxysporum show severe symptoms of dehydration due to the growth of the fungus in vascular tissues. As the disease develops, vascular browning and leaf yellowing can be observed. In the case of more virulent strains, plants die. The pathogen's attack starts with secretion of enzymes degrading the host cell wall. The main aim of the study was to evaluate the role of the cell wall polymers in the flax plant response to the infection in order to better understand the process of resistance and develop new ways to protect plants against infection. For this purpose, the expression of genes involved in cell wall polymer metabolism and corresponding polymer levels were investigated in flax seedlings after incubation with Fusarium oxysporum. RESULTS: This analysis was facilitated by selecting two groups of genes responding differently to the infection. The first group comprised genes strongly affected by the infection and activated later (phenylalanine ammonia lyase and glucosyltransferase). The second group comprised genes which are slightly affected (up to five times) and their expression vary as the infection progresses. Fusarium oxysporum infection did not affect the contents of cell wall polymers, but changed their structure. CONCLUSION: The results suggest that the role of the cell wall polymers in the plant response to Fusarium oxysporum infection is manifested through changes in expression of their genes and rearrangement of the cell wall polymers. Our studies provided new information about the role of cellulose and hemicelluloses in the infection process, the change of their structure and the expression of genes participating in their metabolism during the pathogen infection. We also confirmed the role of pectin and lignin in this process, indicating the major changes at the mRNA level of lignin metabolism genes and the loosening of the pectin structure.

The local treatment and available dressings designed for chronic wounds.

The great diversity of wounds and the broad range of available dressings complicate the selection of proper chronic wound treatment. Choosing the right treatment is the essential step in the healing process. In this review, we focus on chronic nonhealing ulcers, which are a critical problem in clinical practice, and current knowledge about persistent wound care. Here, we present the objectives of local treatment with description of several types of dressings and their ingredients, features, indications, and contraindications. These include hydrocolloid, alginate, hydrogel, and dextranomer dressings; polyurethane foam and membrane dressings; semipermeable polyurethane membrane dressings; and TenderWet (Hartmann, Rock Hill, SC) and flax dressings. There is also a brief section on the use of other alternative wound-healing accelerators, such as platelet-rich plasma and light-emitting diode therapy.

The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene.

BACKGROUND: Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance. METHODS: The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated. RESULTS: The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli. CONCLUSIONS: We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.

Influence of the Bioactive Diet Components on the Gene Expression Regulation.

Diet bioactive components, in the concept of nutrigenetics and nutrigenomics, consist of food constituents, which can transfer information from the external environment and influence gene expression in the cell and thus the function of the whole organism. It is crucial to regard food not only as the source of energy and basic nutriments, crucial for living and organism development, but also as the factor influencing health/disease, biochemical mechanisms, and activation of biochemical pathways. Bioactive components of the diet regulate gene expression through changes in the chromatin structure (including DNA methylation and histone modification), non-coding RNA, activation of transcription factors by signalling cascades, or direct ligand binding to the nuclear receptors. Analysis of interactions between diet components and human genome structure and gene activity is a modern approach that will help to better understand these relations and will allow designing dietary guidances, which can help maintain good health.

Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family.

Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5-9), and have high molecular weights. Batch affinity chromatography on papain-agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC-MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp.

Therapeutic Strategies of Plant-derived Compounds for Diabetes Via Regulation of Monocyte Chemoattractant Protein-1.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family that plays a key role in the inflammatory process. It has been broadly studied in the aspect of its role in obesity and diabetes related diseases. MCP-1 causes the infiltration of macrophages into obese adipose tissue via binding to the CCR2 receptor and is involved in the development of insulin resistance. METHODS: We reviewed the available literature regarding the importance of plant metabolites that regulate MCP-1 activity and are used in the treatment of diabetic disorders. The characteristics of screened papers were described and the important findings were included in this review. RESULTS: This mini-review provides a summary of functions and therapeutic strategies of this chemokine, with a special focus on plant-derived compounds that possess a putative antidiabetic function via a mechanism of MCP-1 interaction. The highlights of this review include the roles of MCP-1 in development of diabetes, the evaluation of plant metabolites that specifically or non-specifically inhibit MCP-1 overproduction, and the molecular mechanisms of this activity. Among these metabolites, we particularly focused on phenolic acids and their derivatives, flavonoids, stilbenes, anthocyanins, capsaicin, alkaloids, plant sterols, terpenes, saponins, unsaturated fatty acids and plant-derived extracts. CONCLUSION: Regarding the increasing number of diabetic patients yearly, the recent progress in the putative therapies needs to be summarized. This article underlines the significance and involvement of the chemokine MCP-1 in the development of obesity, type 2 diabetes, and diabetic complications, with an emphasis on the role of plant metabolites in the regulation of this chemokine and thus the role in the prevention or therapy of diabetes. We suggest that MCP-1 might be a molecular marker of type 2 diabetes.

Optimization of Phenolic Compounds Extraction from Flax Shives and Their Effect on Human Fibroblasts.

The goal of this study was to evaluate the most effective technique for extraction of phenolics present in flax shives and to assess their effect on human fibroblasts. Flax shives are by-products of fibre separation, but they were found to be a rich source of phenolic compounds and thus might have application potential. It was found that the optimal procedure for extraction of phenolics was hydrolysis enhanced by the ultrasound with NaOH for 24 h at 65°C and subsequent extraction with ethyl acetate. The influence of the flax shives extract on fibroblast growth and viability was assessed using the MTT and SRB tests. Moreover, the influence of flax shives extract on the extracellular matrix remodelling process was verified. The 20% increase of the viability was observed upon flax shives extract treatment and the decrease of mRNA collagen genes, an increase of matrix metalloproteinase gene expression, and reduction in levels of interleukin 6, interleukin 10, and suppressor of cytokinin signaling 1 mRNA were observed. Alterations in MCP-1 mRNA levels were dependent on flax shives extract concentration. Thus, we suggested the possible application of flax shives extract in the wound healing process.

Flavonoid C-glucosides Derived from Flax Straw Extracts Reduce Human Breast Cancer Cell Growth In vitro and Induce Apoptosis.

Flax straw of flax varieties that are grown for oil production is a by product which represents a considerable biomass source. Therefore, its potential application for human use is of high interest. Our research has revealed that flax straw is rich in flavonoid C-glucosides, including vitexin, orientin, and isoorientin. The objective of this study was to evaluate the cytotoxicity and possible proapoptotic effect of flax straw derived C-glucosides of flavonoids in the human breast adenocarcinoma cell line (MCF-7). The effects of flax straw derived flavonoid C-glucosides on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B assays. The expression of apoptosis-related genes was assessed by real-time PCR. Our data revealed that flax C-glucosides as well as pure compounds are cytotoxic toward MCF-7 cells and inhibit their proliferation. Moreover, the induction of apoptosis was correlated with the changes in the mRNA level of pro-apoptotic genes. Increased expression of bax and caspase-7, -8, and -9 and decreased mRNA expression of bcl-2 was observed, whereas the mRNA levels of p53 and mdm2 were not altered. These results clearly demonstrated that flax straw metabolites effectively induced growth inhibition and apoptosis in human breast adenocarcinoma cells.

Genes of phenylpropanoid pathway are activated in early response to Fusarium attack in flax plants.

Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant death. To protect itself, the plant activates a number of genes and metabolic pathways, both to counteract the effects of the pathogen, and to eliminate the threat. The identification of the plant genes which respond to infection is the approach, that has been used in this study. Forty-seven flax genes have been identified by means of cDNA subtraction method as those, which respond to pathogen infection. Subtracted genes were classified into several classes and the prevalence of the genes involved in the broad spectrum of antioxidants biosynthesis has been noticed. By means of semi-quantitative RT-PCR and metabolite profiling, the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively verified. We identified the key genes of the synthesis of these compounds. At the same time we determined the level of the metabolites produced in the phenylpropanoid pathway (flavonoids, phenolic acids) in early response to Fusarium attack by means of GC-MS technique. To the best of our knowledge this is the first report to describe genes and metabolites of early flax response to pathogens studied in a comprehensive way.

New biocomposites based on bioplastic flax fibers and biodegradable polymers.

A new generation of entirely biodegradable and bioactive composites with polylactic acid (PLA) or poly-ε-caprolactone (PCL) as the matrix and bioplastic flax fibers as reinforcement were analyzed. Bioplastic fibers contain polyhydroxybutyrate and were obtained from transgenic flax. Biochemical analysis of fibers revealed presence of several antioxidative compounds of hydrophilic (phenolics) and hydrophobic [cannabidiol (CBD), lutein] nature, indicating their high antioxidant potential. The presence of CBD and lutein in flax fibers is reported for the first time. FTIR analysis showed intermolecular hydrogen bonds between the constituents in composite PLA+flax fibers which were not detected in PCL-based composite. Mechanical analysis of prepared composites revealed improved stiffness and a decrease in tensile strength. The viability of human dermal fibroblasts on the surface of composites made of PLA and transgenic flax fibers was the same as for cells cultured without composites and only slightly lower (to 9%) for PCL-based composites. The amount of platelets and Escherichia coli cells aggregated on the surface of the PLA based composites was significantly lower than for pure polymer. Thus, composites made of PLA and transgenic flax fibers seem to have bacteriostatic, platelet anti-aggregated, and non-cytotoxic effect.

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection.

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.