Calcineurin role in porcine oocyte activation.

Calcineurin is required for oocyte exit from meiotic block in metaphase II (MII) stage in invertebrates and also in lower vertebrates. However, the role of calcineurin in mammalian oocyte activation is still unclear. The aim of this study was to determine whether calcineurin is involved in the processes regulating porcine oocyte activation. Indirect immunofluorescence demonstrated localization of both calcineurin subunits, CnA and CnB, especially in the cortex area of MII oocytes, in vitro fertilized and also parthenogenetically activated oocytes. After activation, the fluorescence intensity of the protein in the cortex area of oocytes remains unchanged; the protein calcineurin in the cytoplasm was recorded mainly around the pronuclei. Treatment of matured oocytes with calcineurin inhibitors, cyclosporin A (CsA) and hymenistatin I (HS-I), followed by activation with calcium ionophore A23187, significantly decreased the rate of activated oocytes compared to oocytes that were treated only with calcium ionophore (Ca-Io), (CsA+Ca-Io 25.0% v. Ca-Io 83.3%; HS-I+Ca-Io 32.5% v. Ca-Io 85.0%). Compared to the control, CsA treatment of matured oocytes followed by activation with Ca-Io did not affect the activity level of metaphase-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in activated oocytes evaluated by kinase activity assay. Simultaneous staining of calcineurin and cortical granule content in matured oocytes showed that calcineurin distributed in the cortical area of the oocyte has not been colocalized with cortical granules content. On the other hand, the calcineurin inhibition before parthenogenetic activation leads to a reduction of the cortical reaction level compared to oocytes that were not treated with CsA (complete exocytosis: CsA+Ca-Io 2.6% v. Ca-Io 83.9%; sum of cortical granule brightness: CsA + Ca-Io 0.69 v. Ca-Io 0.15). Our results showed that calcineurin is involved in the process of pig oocyte activation and cortical granule exocytosis; however this regulation seems to be MPF and MAPK independent.

The production of isoflavonoids in Genista tinctoria L. cell suspension culture after abiotic stressors treatment.

The increased content of isoflavonoids in dry cell suspension and nutrient medium was observed after application of electric current and AgNO3 on Genista tinctoria L. cultures in vitro. The highest content of genistin (1.7 mg g(- 1) DW - dry weight) was measured in the dry cell suspension culture after 30 min elicitation of 10 V and 6 h cultivation and daidzein content (3.5 mg g(- 1) DW) was measured after 60 min elicitation of 5 V and 24 h cultivation. In the case of AgNO3 elicitation, the content of genistin in dry cell suspension culture (0.5 mg g(- 1) DW) was highest after 48 h of AgNO3 treatment and concentration of 5.9 × 10(- 4) mol/L. The AgNO3 concentration of 5.9 × 10(- 4) mol/L was also the most effective combination for daidzein production (0.9 mg g(- 1) DW) after 168 h. The results of this study show that the secondary metabolites could also be released from G. tinctoria L. cells into the nutrient medium.

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs.

Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 µM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 µM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP-DPAM, SNAP-OAG and SNAP-PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 µM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP-OAG 0, SNAP-DPAM 17.4 ± 2.5% and SNAP-PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca2+ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP-DPAM 20.7 ± 2.6%, SNAP-OAG 16.7 ± 2.9% or SNAP-PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.

Pyrethroids cypermethrin, deltamethrin and fenvalerate have different effects on in vitro maturation of pig oocytes at different stages of growth.

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate - as potent specific inhibitors of protein phosphatase calcineurin - can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin - cyclosporin A or hymenistatin I - on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin - allethrin or permethrin - had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.

Nitric oxide and meiotic competence of porcine oocytes.

Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor l-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.

Fagopyrum esculentum in vitro.

Common buckwheat is a valuable source of the flavonoid rutin. Its in vitro culture was derived from a young seedling plant and the content of flavonoids in the callus culture was compared with their content in an intact plant. The optimal nutrient medium for the cultivation of the in vitro culture and for the production of flavonoids is Murashige and Skoog medium which contains, during the cultivation in the normal light regime, the growth regulators combination of 2,4-D 1 mg/l and kinetin 1 mg/l.

[Genista tinctoria in vitro]. / Genista tinctoria in vitro.

The paper deals with the effects of different growth regulators on the growth of the culture and production of isoflavonoids in the cultivation of the callus culture Genista tinctoria on MS medium under varying light regimens. Of the regulators tested, the highest growth of the culture was found when using BAP 10 mg/l under continuous lighting. With the use of BAP 0.1 mg/l and under normal light regimen, the production of genistine and daidzenin was even higher than in the intact plant. Of the five isoflavonoids determined, four were demonstrated in the callus culture: genistine, genistein, daidzein, and formononetin. In the intact plant the authors managed to determine daidzein, genistein, and biochanin A.

[Effect of AgNO3 on the production of flavonoids by the culture of Ononis arvensis L. in vitro]. / Vliv AgNO3 na produkci flavonoidu kulturou Ononis arvensis L. in vitro.

The study tested the effect of abiotic elicitor, AgNO3, in different concentrations, on the production of flavonoids in the callus culture Ononis arvensis L. The use of this abiotic elicitor proved to be good to increase the production of flavonoids in in vitro culture. The maximal production was achieved after a 24-hour elicitation with AgNO3 in a concentration c1 (0.5 mg/1)--an increase by 934% versus the control (without the elicitor's action).

[Effect of ultrasound on the production of flavonoids by the culture of Ononis arvensis L. in vitro]. / Vliv ultrazvuku na produkci flavonoido kulturou Ononis arvensis L. in vitro.

The paper tested the effect of ultrasound of output density of 0.1 W/cm3 (stable frequency, 35 kHz) on the production of flavonoids in the suspension culture of Ononis arvensis L. The use of this abiotic elicitor was proved good to increase the production of secondary substances (flavonoids) in the culture of Ononis arvensis L. in vitro. The maximal production of flavonoids began after a 1 min action of ultrasound and immediate withdrawal of the culture--an increase by 677% in comparison with the control (without the action of ultrasound).

[Effect of vanadyl sulfate (VOSO4) on the production of flavonoids by the culture Ononis arvensis L. in vitro]. / Vliv síranu vanadylu (VOSO4) na produkci flavonoidu kultuf Ononis arvensis L. in vitro.

The study tested the effect of vanadyl (IV) sulfate (VOSO4) on the production of flavonoids in the callus and suspension culture of Ononis anrensis L. (Fabaceae). This substance proved to be a good abiotic elicitor for an increase in the production of secondary substances (flavonoids) in the culture of Ononis arvensis L. in vitro. The maximal increase in the formation of flavonoids took place in the callus culture after 24-hour elicitation with VOSO4 in a concentration of 1.227 .10-4 mol/l by 313% in comparison with the control and in the suspension culture in a concentration of 1.227 .10-6 mol/l after 48-hour action of the elicitor by 485% in comparison with the control.

[Silybum marianum in vitro]. / Silybum marianum in vitro.

Callus and suspension cultures of Silybum marianum were derived and the growth and production characteristics were assayed. The maximal production of flavonolignans was achieved at the beginning of cultivation, i.e. on day 10 after inoculation in MS medium with alpha-NAA and on day 15 after inoculation in MS medium with 2.4-D with continuous growth of the culture. The content of silymarin in the plant drug was 3.05%, in the in vitro culture it was just tenths of percents. TLC analysis revealed that the spectrum of flavonolignans of the drug differed from the spectrum of flavonolignans produced by tissue cultures. No differences in the spectra of flavonolignans produced by the callus and suspension cultures were observed. The test for antioxidative activity against the DPPH-radical demonstrated marked antioxidative properties of substances produced by the culture of Silybum marianum.

[Oxidative determination of rutin in tablets using flow injection analysis and chemiluminescence]. / Oxidacní stanovení rutinu v tabletách metodou prutokové injekcní analýzy s chemiluminiscencní detekcí.

A simple, rapid and fully automated flow injection method (FIA) with chemiluminescence detection after oxidation with KMnO4 in acid medium at room temperature has been developed for the determination of routine in pharmaceutical formulation. Hexamethaphosphate sodium was utilized as enhancer of chemiluminescence. The calibration curve was linear in the range 0.01-0.26 mmol.l-1 with detection limit 0.005 mmol.l-1, RSD 0.46% (n = 10) and a sample throughput of 75.h-1 using a 200 microliters sample volume. The method was used for the determination of routine in mass-produced dosage form: Rutin 250 tablets á 50 mg, (firm Nature's Bounty, USA). The FIA method was statistically compared with the official German Pharmacopoeia method and showed comparable accuracy, but with the advantages of simplicity, speed and amounts of reagents consumed.

[Iodoacetic acid as a potential elicitor of increased production of flavonoids in a culture of Ononis arvensis L. in vitro]. / Kyselina jodoctová jako potenciální elicitor zvýsené tvorby flavonoidu v kulture Ononis arvensis L. in vitro.

The paper tested the effect of iodoacetic acid in four different concentrations on the production of flavonoids in the callus and suspension culture of Ononis arvensis L. The elicitor was in contact with the culture for a period of 6; 24; 47; 72, and 168 hours. The obtained results show a statistically significant increase in the content of flavonoids in the suspension culture in comparison with the control in the use of all tested concentrations of the elicitor after 6; 24; 48, and 72 hours of elicitation. The maximal increase by 586% in the production of flavonoids in the suspension culture took place with the use of iodoacetic acid in a concentration of 1 mg/l. In the callus culture, the maximal increase in the formation of flavonoids occurred with the use iodoacetic acid in a concentration of 10 mg/l after 24 hours of elicitation, the content of flavonoids being increased by 529% in comparison with the control.

[Ononis arvensis in vitro--abiotic elicitation]. / Ononis arvensis in vitro--abiotická elicitace.

The effect of two abiotic elicitors--two newly synthesized compounds (substituted anilides of pyrazine-2-carboxylic acids)--on the formation of flavonoids in the callus culture of Ononis arvensis L. was tested. The compounds markedly influenced the production of flavonoids in an in vitro culture. Particularly after elicitation with a solution of compound No. 1 (4-hydroxyanilide 6-chloro-5-terc.butylpyrazine-2-carboxylic acid) in a concentration of 3.32.10(-7) mol.l-1 and within 48 hours of elicitation an increase in flavonoid formation by 976% versus the control took place.

[Effect of jasminic acid on production of flavonoids in a culture of Ononis arvensis L. in vitro]. / Vliv kyseliny jasmínové na produkci flavonoidu kulturou jehlice rolní--Ononis arvensis L. in vitro.

The present paper examined the effect of jasminic acid in three different concentrations (1000 mg/l; 10 mg/l, and 0.1 mg/l) on the formation of flavonoids in the culture Ononis arvensis L. in vitro. The tested concentrations of jasminic acid were applied to the callus and suspension cultures of Ononis arvensis L. in the 4th week of culture growth and elicitation took place for 12; 24; 48; 72, and 168 hours. A statistically significant increase in the content of flavonoids was found in all administered solutions of jasminic acid after 12 hours of elicitation, excepting the concentration c2 (10 mg/l) in the suspension culture. The highest yield was a 55% increase in the content of flavonoids after elicitation with jasminic acid in the concentration c1 (1000 mg/l) in the callus culture.

[Effect of CrCl3 on the in vitro production of flavonoids in a culture of Ononis arvensis L]. / Vliv tvorby flavonoidu v kulture Ononis arvensis L. in vitro pusobením CrCl3.

The paper investigated the effect of the abiotic elicitor chromium trichloride on the production of flavonoids in the callus and suspension cultures of Ononis arvensis L. after 12; 24; 48; 72, and 168-hour administration. The tested elicitor positively influenced the formation of flavonoids. A statistically significant increase in the flavonoid production in the callus culture was recorded with the use of a chromium trichloride solution in the concentration c1 (6.32 x 10(-4) mol/l) after 24; 48; 72, and 168 hours, in the concentration c2 (6.32 x 10(-6) mol/l) after 24; 48, and 72 hours, and in the concentration c3 (6.32 x 10(-8) mol/l) after 12; 24, and 48 hours. In the suspension culture, the flavonoid production was significantly increased by the concentration c1 (6.32 x 10(-4) mol/l) after 12 and 72 hours, the concentration c2 (6.32 x 10(-6) mol/l) after 12; 24; 48, and 72 hours, and the concentration c3 (6.32 x 10(-8) mol/l) after 12; 24; 48, and 72 hours. The maximal increase in the flavonoid production by 98% took place in the callus culture after elicitation with a chromium trichloride in the concentration c3 (6.32 x 10(-8) mol/l) after 48 hours of administration, and in the suspension culture after elicitation with CrCl3 in the concentration c2 (6.32 x 10(-6) mol/l) after 12 hours by 100%.

[Effect of linoleic acid on the production of secondary metabolites]. / Vliv kyseliny linolové na produkci sekundárních metabolitu.

The paper examines the effect of linoleic acid in various concentrations on the production of flavonoids in an Ononis arvensis culture in vitro and the production of anthracene derivatives in a Rheum palmatum culture in vitro. The content of the metabolites under study after elicitation with linoleic acid, which is the precursor of jasminic acid intervening into the course of elicitation, was examined after 12; 24; 48 and 168 hours. The maximal increase in the content of flavonoids (118%) was found after the application of linoleic acid in a concentration of 2 mg/ml after 48 hrs. In concentrations of 0.01; 0.20, and 1 mg/ml after 48 hours of application of the elicitor, the highest increase in the content of flavonoids was observed after 24 hrs in a concentration of 2.0 mg/ml. In the elicitation of the Rheum palmatum culture, the highest response (100%) in the production of anthracene derivatives was observed in linoleic acid in a concentration of 1 mg/ml.

[Interactions between herbal medicines and drugs]. / Interakce mezi lécivými rostlinami a lécivy.

At present the use of medicaments of plant origin is on the increase. It is therefore necessary to take into consideration that there exist known as well as potential interactions between the medicament of the medicinal plant. The problematic plants include Echinacea, Allium cepa, Gingko biloba, Panax ginseng, as well as Hypericum perforatum, Valeriana officinalis, or Glycyrrhiza glabra. Its use should be limited, or completely excluded in the cases of simultaneous therapy with, e.g., warfarin, hepatotoxically acting medicaments, MAOI inhibitors, phenelzin sulphate, or phenytoin, as they may decrease of completely eliminate the therapeutic effect of the administered drugs, or they may cause a toxic damage to the organism.

[Allergic contact dermatitis caused by propolis]. / Alergická kontaktní dermatitis z propolisu.

The present paper describes several individual cases of contact dermatitis in patients using propolis as a component of various cosmetic products, listing the most frequently sensitizing constituents of propolis. It also reports the existence of a cross-reaction between the components of Peruvian balsam and propolis constituents.

[The effect of elicitors from Pseudomonas aeruginosa on the production of flavonoids in cultures of Ononis arvensis L]. / Vliv elicitoru z Pseudomonas aeruginosa na produkci flavonoidu kulturou jehlice rolní--Ononis arvensis L. in vitro.

The effect of the elicitor, killed cells of Pseudomonas aeruginosa, on the production of flavonoids by a callus and a suspension culture of Ononis arvensis L. was examined. The tested elicitor influenced the flavonoid production both in the callus and suspension culture. An increased content of flavonoids in the callus culture was observed with the use of the elicitor in concentrations I and II (5.81 x 10(-2) and 5.81 x 10(-4) g/l). The maximal production took place after a seven-day elicitation with concentration II. This production was higher by 83% as compared with the control. In suspension cultures an increased production was observed in concentrations I and III (5.81 x 10(-2) and 5.81 x 10(6) g/l). The maximal content of flavonoids was measured after 48-hour elicitation with concentration I. In comparison with the control the increase in the flavonoid content was higher by up to 125%.