Pet chinchillas (Chinchilla lanigera): Source of zoonotic Giardia intestinalis, Cryptosporidium ubiquitum and microsporidia of the genera Encephalitozoon and Enterocytozoon.

The domestic chinchilla (Chinchilla lanigera) is kept as a pet and previous studies suggest that it may play an important role as a source of zoonotic parasites, including Giardia intestinalis, Cryptosporidium spp. and microsporidia. In this study, we examined the occurrence and genetic diversity of above mentioned parasites in pet chinchillas in the Czech Republic by PCR/sequencing of the 18S rRNA, TPI, and ITS genes. Of 149 chinchillas from 24 breeders, 91.3 % were positive for G. intestinalis, 8.1 % for Cryptosporidium spp., 2.0 % for Encephalitozoon spp., and 5.4 % for E. bieneusi. Molecular analyses revealed presence of G. intestinalis assemblage B, C. ubiquitum (XIIa family), E. bieneusi genotypes D, SCF2, and, CHN-F1, and E. intestinalis. The infection intensity of G. intestinalis determined by qRT-PCR reached up to 53,978 CPG, C. ubiquitum up to 1409 OPG, E. intestinalis up to 1124 SPG, and E. bieneusi up to 1373 SPG. Only two chinchillas with C. ubiquitum and five with G. intestinalis had diarrhoea at the time of the screening. Three chinchillas in the long-term study were consistently positive for G. intestinalis, with intermittent excretion of C. ubiquitum, E. intestinalis, and E. bieneusi over 25 weeks. The findings indicate that chinchillas are frequently infected with zoonotic parasitic protists, but that these infections rarely show clinical signs. The lack of visible signs could reduce the vigilance of pet owners when handling their chinchillas, increasing the risk of transmission within breeding groups and possibly to humans.

Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris).

BACKGROUND: Cryptosporidium spp. are globally distributed parasites that infect epithelial cells in the microvillus border of the gastrointestinal tract of all classes of vertebrates. Cryptosporidium chipmunk genotype I is a common parasite in North American tree squirrels. It was introduced into Europe with eastern gray squirrels and poses an infection risk to native European squirrel species, for which infection is fatal. In this study, the biology and genetic variability of different isolates of chipmunk genotype I were investigated. METHODS: The genetic diversity of Cryptosporidium chipmunk genotype I was analyzed by PCR/sequencing of the SSU rRNA, actin, HSP70, COWP, TRAP-C1 and gp60 genes. The biology of chipmunk genotype I, including oocyst size, localization of the life cycle stages and pathology, was examined by light and electron microscopy and histology. Infectivity to Eurasian red squirrels and eastern gray squirrels was verified experimentally. RESULTS: Phylogenic analyses at studied genes revealed that chipmunk genotype I is genetically distinct from other Cryptosporidium spp. No detectable infection occurred in chickens and guinea pigs experimentally inoculated with chipmunk genotype I, while in laboratory mice, ferrets, gerbils, Eurasian red squirrels and eastern gray squirrels, oocyst shedding began between 4 and 11 days post infection. While infection in mice, gerbils, ferrets and eastern gray squirrels was asymptomatic or had mild clinical signs, Eurasian red squirrels developed severe cryptosporidiosis that resulted in host death. The rapid onset of clinical signs characterized by severe diarrhea, apathy, loss of appetite and subsequent death of the individual may explain the sporadic occurrence of this Cryptosporidium in field studies and its concurrent spread in the population of native European squirrels. Oocysts obtained from a naturally infected human, the original inoculum, were 5.64 × 5.37 µm and did not differ in size from oocysts obtained from experimentally infected hosts. Cryptosporidium chipmunk genotype I infection was localized exclusively in the cecum and anterior part of the colon. CONCLUSIONS: Based on these differences in genetics, host specificity and pathogenicity, we propose the name Cryptosporidium mortiferum n. sp. for this parasite previously known as Cryptosporidium chipmunk genotype I.

Astaxanthin Extract from Haematococcus pluvialis and Its Fractions of Astaxanthin Mono- and Diesters Obtained by CCC Show Differential Antioxidant and Cytoprotective Effects on Naïve-Mouse Spleen Cells.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.

Description of Cryptosporidium ornithophilus n. sp. (Apicomplexa: Cryptosporidiidae) in farmed ostriches.

BACKGROUND: Avian cryptosporidiosis is a common parasitic disease that is caused by five species, which are well characterised at the molecular and biological level, and more than 18 genotypes for which we have limited information. In this study, we determined the occurrence and molecular characteristics of Cryptosporidium spp. in farmed ostriches in the Czech Republic. METHODS: The occurrence and genetic identity of Cryptosporidium spp. were analysed by microscopy and PCR/sequencing of the small subunit rRNA, actin, HSP70 and gp60 genes. Cryptosporidium avian genotype II was examined from naturally and experimentally infected hosts and measured using differential interference contrast. The localisation of the life-cycle stages was studied by electron microscopy and histologically. Infectivity of Cryptosporidium avian genotype II for cockatiels (Nymphicus hollandicus (Kerr)), chickens (Gallus gallus f. domestica (L.)), geese (Anser anser f. domestica (L.)), SCID and BALB/c mice (Mus musculus L.) was verified. RESULTS: A total of 204 individual faecal samples were examined for Cryptosporidium spp. using differential staining and PCR/sequencing. Phylogenetic analysis of small subunit rRNA, actin, HSP70 and gp60 gene sequences showed the presence of Cryptosporidium avian genotype II (n = 7) and C. ubiquitum Fayer, Santín & Macarisin, 2010 IXa (n = 5). Only ostriches infected with Cryptosporidium avian genotype II shed oocysts that were detectable by microscopy. Oocysts were purified from a pooled sample of four birds, characterised morphometrically and used in experimental infections to determine biological characteristics. Oocysts of Cryptosporidium avian genotype II measure on average 6.13 × 5.15 µm, and are indistinguishable by size from C. baileyi Current, Upton & Haynes, 1986 and C. avium Holubová, Sak, Horcicková, Hlásková, Kvetonová, Menchaca, McEvoy & Kvác, 2016. Cryptosporidium avian genotype II was experimentally infectious for geese, chickens and cockatiels, with a prepatent period of four, seven and eight days post-infection, respectively. The infection intensity ranged from 1000 to 16,000 oocysts per gram. None of the naturally or experimentally infected birds developed clinical signs in the present study. CONCLUSIONS: The molecular and biological characteristics of Cryptosporidium avian genotype II, described here, support the establishment of a new species, Cryptosporidium ornithophilus n. sp.

A chicken embryo model for the maintenance and amplification of Cryptosporidium parvum and Cryptosporidium baileyi oocysts.

Cryptosporidium is a genus of apicomplexan parasites that inhabit the respiratory and gastrointestinal tracts of vertebrates. Research of these parasites is limited by a lack of model hosts. This study aimed to determine the extent to which infection at the embryo stage can enhance the propagation of Cryptosporidium oocysts in chickens. Nine-day-old chicken embryos and one-day-old chickens were experimentally infected with different doses of Cryptosporidium baileyi and Cryptosporidium parvum oocysts. Post hatching, all chickens had demonstrable infections, and the infection dose had no effect on the course of infection. Chickens infected as embryos shed oocysts immediately after hatching and shed significantly more oocysts over the course of the infection than chickens infected as one-day-olds. In chickens infected as embryos, C. baileyi was found in all organs except the brain whereas, C. parvum was only found in the gastrointestinal tract and trachea. In chickens infected as one-day-olds, C. baileyi was only found in the gastrointestinal tract and trachea. Chickens infected as embryos with C. baileyi died within 16 days of hatching. All other chickens cleared the infection. Infection of chickens as embryos could be used as an effective and simple model for the propagation of C. baileyi and C. parvum.

Vanadium elicitation of Trifolium pratense L. cell culture and possible pathways of produced isoflavones transport across the plasma membrane.

KEY MESSAGE: Vanadium compounds increased the content and release of distinct isoflavones in a Trifolium pratense suspension culture. Regarding transport-mechanism inhibitors, the process was mostly facilitated by ABC proteins and vesicular transport. The transport of isoflavones and other secondary metabolites is an important part of metabolism within plants and cultures in vitro regarding their role in defence against various abiotic and biotic stressors. This research focuses on the way how to increase production and exudation of isoflavones by application of chemical elicitor and the basic identification of their transport mechanisms across cell membranes. The release of five isoflavones (genistin, genistein, biochanin A, daidzein, and formononetin) into a nutrient medium was determined in a Trifolium pratense var. DO-8 suspension culture after two vanadium compound treatments and cultivation for 24 and 48 h. The NH4VO3 solution caused a higher concentration of isoflavones in the medium after 24 h. This increased content of secondary metabolites was subsequently suppressed by distinct transport-mechanism inhibitors. The transport of isoflavones in T. pratense was mostly affected by ABC inhibitors from the multidrug-resistance-associated protein subfamily, but the genistein concentration in the medium was lower after treatment with multidrug-resistance protein subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased the concentration of some isoflavones in the nutrient medium.

Differential Effects of the Flavonolignans Silybin, Silychristin and 2,3-Dehydrosilybin on Mesocestoides vogae Larvae (Cestoda) under Hypoxic and Aerobic In Vitro Conditions.

Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans-silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)-on M. vogae larvae at concentrations of 5 and 50 µM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 µM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 µM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.

Isoflavones Production and Possible Mechanism of Their Exudation in Genista tinctoria L. Suspension Culture after Treatment with Vanadium Compounds.

The family Fabaceae traditionally serves as a food and herbal remedies source. Certain plants serve for treatment of menopausal symptoms based on a presence of typical secondary metabolites, isoflavones. Beside soybean and clovers, other plants or cultures in vitro can produce these molecules. A cultivation in vitro can be enhanced by elicitation that stimulates metabolites biosynthesis via stress reaction. Vanadium compounds have been already described as potential elicitors, and the aim of this study was to determine the impact of NH4VO3 and VOSO4 solutions on isoflavones production in Genista tinctoria L. cell cultures. The significant increase of isoflavones content, such as genistin, genistein, or formononetin, was measured in a nutrient medium or dry mass after NH4VO3 treatment for 24 or 48 h. The possible transport mechanism of isoflavones release as a result of elicitation was further evaluated. An incubation with different transport inhibitors prior to elicitation took effect on isoflavones content in the medium. However, there was a non-ended result for particular metabolites such as genistein and daidzein, where ATP-binding cassette (ABC) or, alternatively, multidrug and toxin extrusion (MATE) proteins can participate. Possible elicitation by some inhibitors was discussed as a result of their pleiotropic effect. Despite this outcome, the determination of the transport mechanism is an important step for identification of the specific transporter.

Drought-tolerant and drought-sensitive genotypes of maize (Zea mays L.) differ in contents of endogenous brassinosteroids and their drought-induced changes.

The contents of endogenous brassinosteroids (BRs) together with various aspects of plant morphology, water management, photosynthesis and protection against cell damage were assessed in two maize genotypes that differed in their drought sensitivity. The presence of 28-norbrassinolide in rather high quantities (1-2 pg mg-1 fresh mass) in the leaves of monocot plants is reported for the first time. The intraspecific variability in the presence/content of the individual BRs in drought-stressed plants is also described for the first time. The drought-resistant genotype was characterised by a significantly higher content of total endogenous BRs (particularly typhasterol and 28-norbrassinolide) compared with the drought-sensitive genotype. On the other hand, the drought-sensitive genotype showed higher levels of 28-norcastasterone. Both genotypes also differed in the drought-induced reduction/elevation of the levels of 28-norbrassinolide, 28-norcastasterone, 28-homocastasterone and 28-homodolichosterone. The differences observed between both genotypes in the endogenous BR content are probably correlated with their different degrees of drought sensitivity, which was demonstrated at various levels of plant morphology, physiology and biochemistry.

Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-Infectives and Abiotic Elicitors.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 µg/mL, 5.19 µM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 µg/mL, 18.91 µM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

Azorella compacta Infusion Activates Human Immune Cells and Scavenges Free Radicals In vitro.

BACKGROUND: Azorella compacta is traditionally used in the form of tea (infusion), in the Andean region of South America, to treat various chronic diseases. However, the health-promoting properties of this herbal tea have not yet been extensively explored. MATERIALS AND METHODS: The free radical scavenging activity of A. compacta infusion (ACI) was evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and superoxide anion radical assays. The activation of immune cells by ACI, as determined by cell surface cluster of differentiation 69 expression, was measured by flow cytometry. The qualitative polyphenolic composition of ACI was investigated by HPLC/PDA/ESI-MS, (High-performance liquid chromatography coupled with photodiode array detection and electrospray ionization - mass spectrometry) and the total content of polyphenols was estimated by spectrophotometric methods. RESULTS: Eight polyphenols including chlorogenic acid, 6,8-di-C-hexosyl apigenin, isoorientin, orientin, dicaffeoylquinic acid, biochanin A-O-glucoside, biochanin A-O-(malonyl)-glucoside, and licoisoflavone A were tentatively identified in ACI. The total contents of phenols, flavonoids, and tannins in lyophilized ACI were 5.40 mg/100 mg ACI, 1.79 mg/100 mg ACI, and 1.76 mg/100 mg ACI, respectively. ACI, within the range of 25-400 µg/mL, scavenged DPPH and O2.- by 15-90% and 20-88%, respectively. The human natural killer (NK) cells were substantially activated by ACI, whereas T cells and granulocytes were slightly stimulated. CONCLUSION: Overall, the results demonstrate the free radical scavenging and immune-stimulating properties of ACI, and support, at least in part, its potential utilization as a functional herbal tea. for preventing chronic diseases and as a nonspecific immune stimulator during human immunosenescence. SUMMARY: The total contents of phenols, flavonoids, and tannins in Azorella compacta infusion (ACI) were 5.40 mg/100 mg ACI, 1.79 mg/100 mg ACI, and 1.76 mg/100 mg ACI, respectively.Eight polyphenols including chlorogenic acid, 6,8-di-C-hexosyl apigenin, isoorientin, orientin, dicaffeoylquinic acid, biochanin A-O-glucoside, biochanin A-O-(malonyl)-glucoside, and licoisoflavone A were tentatively identified in ACI by HPLC/PDA/ESI-MS.ACI, within the range of 25-400 µg/ml, scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH) and O2. by 15-90% and 20-88%, respectively.The human natural killer (NK) cells were substantially activated by ACI, whereas T cells and granulocytes were slightly stimulated. Abbreviations used: ESI: electrospray ionization, HPLC: high performance liquid chromatography, PDA: photodiode array detector, MS: mass spectrometry, MS/MS: tandem mass spectrometry, MW: molecular weight, m/z: mass-to-charge ratio, FITC: fluorescent isothiocyanate, PE: phycoerythrin.

The disadvantages of being a hybrid during drought: A combined analysis of plant morphology, physiology and leaf proteome in maize.

A comparative analysis of various parameters that characterize plant morphology, growth, water status, photosynthesis, cell damage, and antioxidative and osmoprotective systems together with an iTRAQ analysis of the leaf proteome was performed in two inbred lines of maize (Zea mays L.) differing in drought susceptibility and their reciprocal F1 hybrids. The aim of this study was to dissect the parent-hybrid relationships to better understand the mechanisms of the heterotic effect and its potential association with the stress response. The results clearly showed that the four examined genotypes have completely different strategies for coping with limited water availability and that the inherent properties of the F1 hybrids, i.e. positive heterosis in morphological parameters (or, more generally, a larger plant body) becomes a distinct disadvantage when the water supply is limited. However, although a greater loss of photosynthetic efficiency was an inherent disadvantage, the precise causes and consequences of the original predisposition towards faster growth and biomass accumulation differed even between reciprocal hybrids. Both maternal and paternal parents could be imitated by their progeny in some aspects of the drought response (e.g., the absence of general protein down-regulation, changes in the levels of some carbon fixation or other photosynthetic proteins). Nevertheless, other features (e.g., dehydrin or light-harvesting protein contents, reduced chloroplast proteosynthesis) were quite unique to a particular hybrid. Our study also confirmed that the strategy for leaving stomata open even when the water supply is limited (coupled to a smaller body size and some other physiological properties), observed in one of our inbred lines, is associated with drought-resistance not only during mild drought (as we showed previously) but also during more severe drought conditions.

Arbutin Content and Tyrosinase Activity of Bergenia Extracts.

The total arbutin content in the leaves of all the studied Bergenia plants (B. crassifolia, B. ciliata and B. x ornata) was determined. The highest values of the arbutin content have been established for B. crassifolia (58.9 ± 0.7 mg.g-¹ DW) and B. x ornata (51.0 ± 1.21 mg.g-¹ DW), and the lowest for B. ciliata (5.9 ± 0.6 mg.g-¹ DW). Arbutin concentration in the Bergenia leaves was the lowest in spring, in the autumn, on the contrary it increased. All the tested aqueous extracts caused a dose-dependent increase in diphenolase activity of fungal tyrosinase in a similar way as arbutin. On the other hand, all the ethanol extracts inhibited the diphenolase activity of tyrosinase.

Production of Podophyllotoxin by Plant Tissue Cultures of Juniperus virginiana.

Plant tissue cultures are a potential source of secondary metabolites. However, their production, when compared with intact plants, is usually lower. Phenylalanine, a biogenetic precursor of podophyllotoxin, was used to stimulate podophyllotoxin production in callus and suspension cultures of Juniperus virginiana L. The best phenylalanine effect on podophyllotoxin production was manifested in three-years-old callus cultures after a 21-days application of a 10 mmol/L concentration. A podophyllotoxin content of 0.15 mg/g DW was determined, which was about 400% higher in comparison with the control. The maximum content (0.48 mg/g DW) in newly derived suspension cultures (the 4' passage) was induced by 14-days application of a I mmol/L concentration; this was about 243% higher than the control. In one-year-old suspension cultures the highest podophyllotoxin content (0.56 mg/g DW) was recorded also after 14-days application of a I mmol/L concentration; this was about 211% higher than in the control cultures.

Effect of Selected Pyrazine Derivatives on the Production of Phenolics and Rutin in Urtica dioica and Fagopyrum esculentum.

The effect of four pyrazine derivatives on the content of phenolic compounds in Urtica dioica L. and rutin in Fagopyrum esculentum Moench was studied. Pyrazine derivatives H1 and H2 were used on U. dioica, and derivatives S1 and S2 on F. esculentum, both separately and in combination with urea. The content of phenolic compounds in the stems of U. dioica after treatment with H2 at a concentration of 10(-3) M significantly increased compared with the control and to a lower concentration of the same pyrazine derivative. In the case of S1 and S2 for F. esculentum, rutin content also increased in stems, mainly after treatment together with urea. By contrast, rutin and phenolics contents in the leaves did not change in comparison with controls after application of H1, H2, S I and S2. Treatment with H1 and H2 in two chosen concentrations resulted in a significant increase in the net photosynthetic rate, transpiration rate and stomatal conductance. A slight increase in the rate of photosynthesis was observed also after application of variants of S1 and S1 with urea. Pyrazine derivatives did not show any effect on either the relative content of chlorophyll or chlorophyll fluorescence. A slight weight reduction of above ground biomass was shown only after application of Si and S2. Dark necrosis on the edges and center of the leaves was observed in all treated plants after pyrazine application. The results suggest that all the pyrazine derivatives possess herbicidal effects.

New Synthetic Pyrazine Carboxamide Derivatives as Potential Elicitors in Production of Secondary Metabolite in In vitro Cultures.

BACKGROUND: Silymarin, an active polyphenolic fraction of Silybum marianum, and high flavonoid content of Fagopyrum possess various interesting biological activities. The substituted pyrazine-2-carboxamides were previously used as effective elicitors of studied secondary metabolites. OBJECTIVE: To study the effect of new synthetic pyrazine carboxamide derivatives, N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide (1) and 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide (2), on flavonolignan and flavonoid production in S. marianum and Fagopyrumes culentum in vitro cultures. MATERIALS AND METHODS: Callus and suspension cultures were cultured on MS medium containing α-naphtaleneacetic acid or 2,4-D. Three elicitor concentrations for different exposure times were tested. Dried and powdered samples of callus and suspension cultures were extracted with methanol and analyzed by DAD-HPLC. RESULTS: Compound 1 showed as a good elicitor of taxifolin production. The effect on silymarin complex was less visible with a maximum between 24 and 48 h after 3.292 ×10(-4) mol/L concentration. The detailed analysis showed that silychristin was the most abundant. Compound 2 was effective in rutin production only in callus culture with maximum 24 h and 168 h after application of 3.3756 ×10(-3) mol/L concentration and 48 and 72 h after 3.3756 ×10(-4) mol/L concentration. CONCLUSION: From the results of the performed experiments, it can be concluded that compound 1 shows to be suitable elicitor for enhanced production of taxifolin and silychristin in S. marianum, mainly when 3.292 ×10(-4) mol/L concentration was used, and compound 2 is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum. SUMMARY: The influence of two new synthetic pyrazine-2-carboxamidesderivatives on secondary metabolite content of Silybum marianum and Fagopyrum esculentum in vitro cultures was tested.In S. marianum, the derivate N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide showed as a good elicitor of taxifolin production and less effective for silymarin complex production with silychristin as the most abundant.The derivate 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum.

Bergenin Content and Free Radical Scavenging Activity of Bergenia Extracts. .

Our research was focused on the evaluation of bergenin content and free radical scavenging activity of extracts prepared from three different species of Bergenia - B. crassifolia (L.) Fritsch., B. ciliata (Haw.) Sternb. and B. x ornata Stein. collected during different seasons. Using an HPLC method, the highest total amount of bergenin was revealed in the leaves of B. x ornata and B. crassifolia (4.9 - 5.1 mg x g(-1)). Free radical scavenging power was determined by two methods--FRAP and NADH. The best free radical scavengers were B. crassifolia (FRAP: 6.7 - 15.9 mg GAE. 100g(-1); NADH: 20.3 - 50.9%) and B. ornata (FRAP: 13.7 - 15.2 mg GAE. 100g(-1); NADH: 29.3 - 31.1%). The lowest content of bergenin and the weakest radical scavenger was B. ciliata (bergenin: 3.1 mg x g(-1); FRAP: 5.5 - 11.0 mg GAE.100g(-1); NADH: 23.2 - 25.6%). The presence of a large percentage of bergenin is responsible for the radical scavenging activity, as shown by the results from the FRAP and NADH assays. Significant, positive correlation was found between bergenin content and radical scavenging activity in both methods.

Hydrogen sulfide donor protects porcine oocytes against aging and improves the developmental potential of aged porcine oocytes.

Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.

Effect of ultrasound on the isoflavonoid production in Genista tinctoria L. suspension cultures.

BACKGROUND: Application of ultrasound (US) to biotechnology is relatively new but several processes that take place in the presence of cells or enzymes are activated by ultrasonic waves. Genista tinctoria L. (Fabaceae) is rich on various kind of flavonoids, including isoflavones with valuable estrogenic activity. OBJECTIVE: This study verified use of low-energy US elicitor to enhance secondary metabolite production in plant cell cultures. MATERIALS AND METHODS: Suspension cultures of G. tinctoria cells was exposed to low-power US (with fixed frequency 35 kHz and power level 0.1 mW/cm(3)) for period 1-5 min. RESULTS: The US exposure significantly stimulated genistin content (0.8 mg/g DW) after 3 min of US treatment (sampled after 72 h). The highest daidzein level (1.4 mg/g DW) was reached after US irradiation for 5 min and 168 h sampling. CONCLUSION: The achieved results suggest that US can act as a potent abiotic elicitor to induce the defense responses of plant cells and to stimulate secondary metabolite production in plant cell cultures.

Antioxidant activity and phenolic content of Bergenia crassifolia, B. x ornata and B. ciliata.

This study focused on a phytochemical analysis of Bergenia crassifolia (L.) Fritsch., B. ciliata (Haw.) Sternb., and B. x ornata Stein. and evaluation of their free radical scavenging properties. Arbutin and total tannin contents of the leaves of the Bergenia species were determined during different seasons. The present study also aimed at analyzing, for the first time, environmental influence on concentrations of phenolic metabolites in Bergenia leaves. The highest total tannin content was found in the leaves of B. crassifolia (24.9-48.7 mg x g(-1) DW) and B. x ornata (36.9 mg.g(-1) DW). The highest amount of arbutin was in the leaves of B. x ornata (35.8-51.0 mg.g(-1) DW) and B. crassifolia (24.6-41.7 mg x g(-1) DW). Autumn was better than spring for the collection of Bergenia leaves for the highest amount of arbutin (B. x ornata: 51.0 mg x g(-1) DW). Free radical scavenging potential, in DPPH and ABTS assays, of the water leaf extracts revealed that extracts of B. crassifolia and B. x ornata are the most active radical scavengers. Antioxidant activity correlated well with the content of total tannin, especially in the ABTS assay, which suggests an important role for these compounds in antioxidant activity. It was shown that phenolic concentrations in Bergenia leaves are affected by seasonal factors. A significant correlation was found between arbutin and tannin contents and the average humidity.