Mitochondrial activity of 2,6-diaminopurine in Saccharomyces cerevisiae.

Summary 2,6-diaminpurine (DAP) selectively inhibited mitochondrial protein synthesis in yeast cells with concomitant failure of cells to grow in non-fermentable (yeast extract, glycerol) medium. The selectivity was pronounced in all strains tested (15) nearly all of which were able to grow in yeast extract, glucose medium containing 5 mg/ml DAP (maximum solubility) whereas growth was arrested in all strains at 250–500 g/ml DAP in the glycerol medium. The inhibition was reversed by further addition of adenine to the culture medium. RNA synthesis in rat liver mitochondria was depressed by DAP suggesting that the analogue affected RNA polymerase activity.There was no evidence of nuclear mutagenicity by DAP but resistance to the antibiotics chloramphenicol and oligomycin was induced by the drug. Genetic evidence, although limited, indicated that the resistance mutations were cytoplasmic. The mitochondrial petite mutation was also induced by DAP but only at comparatively high concentrations. The mutagenic effects were seen only in the glycerol medium.     

Mitochondrial genetic damage induced in yeast by a photoactivated furocoumarin in combination with ethidium bromide or ultraviolet light

Summary Ethidium bromide (EB) and ultraviolet light (UV) in combination are known to produce a synergistic induction of petite mutants in yeast. Two other agents were combined with EB, 3-Carbethoxypsoralene (3 CPs) activated by 365 nm light or rays. EB in combination with 3 CPs also resulted in an enhanced production of petite mutants. After the photoaddition of 3 CPs in exponential phase cells, recovery of the petite mutation during dark liquid holding was inhibited by the presence of EB producing an enhanced number of petite mutants. The behavior of mitochondrial antibiotic resistance markers after individual and combined treatments with EB and 3 CPs indicates a random loss of markers after EB and a preferential loss of a certain region for the 3 CPs photoaddition. The combination of the two agents leads to an additivity of total drug marker losses rather than a synergistic loss. The combination of EB with rays produced no enhancement in petite induction. A combination of UV and 3 CPs showed a synergistic interaction for petite induction. These results indicate that the three agents, EB, UV and 3 CPs photoaddition may share a common repair step for mitochondrial lesions.     

Mitochondrial membranes and mutagenesis by ethidium bromide

The conversion of wild type (ρ⁺) to cytoplasmic petites (ρ^?) in Saccharomyces cerevisiae, à mutation in mitochondrial DNA, can be brought about with high efficiency by low concentrations of ethidium bromide (EB). The rate and extent of mutagenesis and its expression can be influenced, and even reversed, by a number of genetic lesions, agents or treatments affecting mitochondrial structure and metabolism. Among them are incubation at 45°, exposure to Antimycin A, growth on different carbon sources and the presence or absence of 2 different gene products previously implicated in the repair of UV induced lesions in mitochondrial DNA. Based on these observations a model for EB mutagenesis is advanced which postulates a complex between mitochondrial DNA and the inner membrane as the target susceptible to modification by EB. This model predicts that altered membranes should lead to changes in the susceptibility of cells to the mutagenic action of EB. This prediction has been verified by comparing cells that contain one of 2 structurally quite distinct monounsaturated C₁₈ fatty acids in their mitochondrial phospholipids: greater resistance to mutagenesis and ease of thermal protection is exhibited when cells – and mitochondria – contain oleic (Δ⁹cis, m.p. < 5°) rather than petroselinic (Δ⁶cis, m.p. 28°) acid in their phospholipids. As a corollary, studies on EB mutagenesis and mitochondrial DNA may be used as probes for the mitochondrial inner membrane to reveal some perhaps novel functions.     

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

Mitochondrial genetics

Summary Cytoplasmic petite mutants of Saccharomyces cerevisiae carrying the gene conferring the resistance to chloramphenicol on one hand and the gene conferring the resistance to erythromycin on the other, have been crossed with each other. The two types of petites differed in the buoyant densities of their mitochondrial DNA. A novel type of evidence has been adduced, that the two genes are indeed located on mitochondrial DNA. Diploid petite recombinants were found, carrying both genes and containing not a mixture of the two parental DNAs but a new species of mitochondrial DNA of intermediate buoyant density. Recombination of mitochondrial genes involves therefore breakage and reunion of DNA molecules. New suppressiveness, different from the two parental ones, can result from the recombination of mitochondrial DNA. Recombination between petite mutants implies that the mitochondrial recombination enzymes have to be synthesized on cytosol ribosomes.     

Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Adriamycin (ADR)‐resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK‐8 Assay was performed to evaluate the cytotoxicity of anti‐tumor drugs; BCECF‐AM pH‐sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H⁺‐ATPases (V‐ATPases), mTOR, HIF‐1α, P‐glycoprotein (P‐gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose‐dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose‐dependently. After 24‐h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non‐PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V‐ATPases, mTOR, HIF‐1α, P‐gp, and MRP1, and alter intracellular expressions in parent and ADR‐resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti‐tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti‐tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V‐ATPases/mTOR/HIF‐1α/P‐gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 2474–2487, 2012. © 2012 Wiley Periodicals, Inc.     

The size of mitochondrial DNA from a cytoplasmic petite mutant of Saccharomyces cerevisiae

Summary Mitochondrial DNA has been isolated from a cytoplasmic petite mutant of Saccharomyces cerevisiae which has retained only about 2% of the mitochondrial wild type genome. The denatured DNA was analyzed by agarose gel electrophoresis and a homogeneous, single band of DNA was found. Petite and wild type mitochondrial DNAs exhibited similar gel electrophoretic mobilities. Using denatured DNA from the E. coli phages T4 and T3 for comparison a molecular weight of 55×10⁶ daltons has been calculated for the double-stranded petite mitochondrial DNA. On the basis of this observation most of the mitochondrial DNA of this petite mutant appeared to consist of a polymer of about 50 repeats to account for a size similar to that of the wild type molecule. Thus a regulatory mechanism might exist which keeps constant the physical size of the mitochondrial DNA molecule in spite of the elimination of large fractions of the wild type genome.Dedicated to Dr. Dr. h. c. Peter Michaelis on the occasion of his 75th birthday     

Inhibition of mitochondrial protein synthesis in vivo by erythromycin in Schizosaccharomyces pombe and Saccharomyces cerevisiae

Summary In the presence of erythromycin (0.01 mg/ml) growth of Schizosaccharomyces pombe in non-fermentable substrate (glycerol) is reduced to 5–15% of the control without erythromycin, whereas growth in fermentable substrate (5% glucose) is left unaffected by concentrations up to 5 mg/ml. The reduction of growth under derepressed conditions is paralleled by inhibition of the formation of cytochromes a·a₃ and b. Mitochondrial protein synthesis is inhibited to about 50% in Schizosaccharomyces pombe and to about 90% in Saccharomyces cerevisiae. These results support the hypothesis that inhibition of mitochondrial protein synthesis is the primary effect of erythromycin.     

Toxicity of 4-Nitroquinoline 1-Oxide for Crithidia fasciculata

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cysteine, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

Isolation of metabolically active Petite mutants of Kluyveromyces lactis, a petite-negative yeast

Summary Conditions under which complete cultures of the petite-negative yeast Kluyveromyces lactis can be converted to metabolically active petite mutants have been found. These mutants, which lack mitochondrial protein synthesis have been shown to be metabolically active by their ability to exclude the dye trypan blue. They appear to possess a functional protein synthesising system, which is sensitive to the inhibitor trichodermin. However, on transfer to solid nutrient medium, these mutants fail to grow normally, and give rise to microcolonies composed of up to a thousand cells. These colonies autolyse after several days.     

Effect of zearalenone and estradiol benzoate on serum concentrations of LH, FSH and prolactin in ovariectomized gilts

Six ovariectomized gilts were given zearalenone (Z), estradiol benzoate (EB) or vehicle in a replicated 3 x 3 Latin square design. Zearalenone was added to 2.3 kg of a corn-soybean ration at a dose of 1 mg Z/kg body weight; EB was given intramuscularly at 0.1 mg EB/kg body weight. Control gilts received vehicle solvent for both Z and EB. Blood samples were collected from indwelling jugular cannulas at 6-h intervals for 48 h before Z, EB or vehicle was given. After treatment, blood samples were drawn at 6-h intervals for an additional 84 h. Serum concentrations of luteinizing hormone (LH) decreased (P<0.001) from 4.67 ng/ml to 0.29 ng/ml within 6 h of EB. From 54 to 84 h after EB, serum concentrations of LH rose to 15.60 ng/ml (P<0.001). Serum concentrations of LH were reduced (P<0.001) in a similar pattern after Z (3.70 ng/ml to 0.49 ng/ml), but a rise in serum LH was not observed 54 to 84 h after Z (1.30 ng/ml). Serum concentrations of LH remained unchanged (P=0.55) in gilts given vehicle. Serum concentrations of follicle stimulating hormone (FSH) were suppressed (P<0.03) at 6 h in EB (19.10 vs 11.35 ng/ml) and Z gilts (16.16 vs 11.41 ng/ml) but remained unchanged in vehicle gilts. Serum concentrations of FSH did not change in EB or Z gilts during the next 36 h. These data indicate that the suppressive action of Z on serum concentrations of LH and FSH was similar to that of EB, while the biphasic stimulatory effect of EB for LH was not manifested by Z.     

In vitro evaluation of six fungicides on radial mycelial growth and regrowth of Fusarium pallidoroseum isolated from castor (Ricinus communis) in Samaru,Nigeria

Six fungicides at three rates (1.5x, 1.0x and 0.5x mg a.i/ml) were evaluated on radial growth and and regrowth of mycelia of Fusarium pallidoroseum isolated from castor (Ricinus communis) in vitro. It was observed that the fungicides (Benomyl, Benomyl + Thiram, Mancozeb, Metalaxyl-m + Thiomethoxan + Difenconazol, Tricyclazole and Carbendazim + Mancozeb) at all the concentrations tested inhibited mycelial growth and regrowth of the fungus. Benomyl, Benomyl + Thiram and Tricyclazole completely inhibited mycelia growth of fungus at 1.5x, 1.0x and 0.5x mg a.i/ml. Metalaxyl-m + Thiomethoxan + Difenconazole, Carbendazim + Mancozeb partially inhibited radial growth and re-growth of mycelia only at 1.5x mg a.i/ml, not at 1.0x and 0.5x mg a.i/ml. The inhibitory effect of all the fungicides on mycelia growth and re-growth was greatest at 1.x5 mg a.i/ml.     

Growth of eukaryotic cells in relation to the structure of mitochondrial membranes and mitochondrial genome

Viability ofpetite-negative yeast, such asKluyveromyces lactis, is dependent on functional mitochondrial genome encoding essential components of both mitochondrial protein synthesizing system and oxidative phosphorylation. We have isolated several nuclear mutants impaired in mitochondrial functions that were unable to grow on non-fermentable carbon and energy sources. They were used for the isolation and molecular characterization of the three genes encoding apocytochromec, apocytochromec ₁ and the protein involved in the biogenesis of cytochrome oxidase. All cytochrome-deficient mutants were viable and did not survive the ethidium bromide mutagenesis.Petite-positiveSaccharomyces cerevisiae requires intact mitochondrial genome when its phosphatidylglycerolphosphate synthase was inactivated due to mutation in thePEL1 gene. UsingPEL-lacZ fusion genes it was demonstrated that Pel1p is a mitochondrial protein (expressed in response tomyo-inositol and choline). Thepel1 mutant was deficient in phosphatidylglycerol (PG) and cardiolipin (CL) and itsrho ⁻/rho ⁰ mutants grew extremely slowly on complex medium with glucose. Under the same conditions the growth rate of thecrd1 rho ⁻ double mutants was similar to that of its parentcrd1 mutant deficient in cardiolipin synthase and accumulating PG. The results demonstrate that thepetite negativity in yeast is not dependent on an intact respiratory chain or functional oxidative phosphorylation. The presence of the negatively charged PG or CL seems to be essential for the maintenance of specific mitochondrial functions required for the normal mitotic growth of yeast cells. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

CORRELATION BETWEEN ELECTRICAL ACTIVITY AND SPLITTING OF PHOSPHOLIPIDS BY SNAKE VENOM IN THE SINGLE ELECTROPLAX

Abstract— Cottonmouth moccasin snake venom (SV) was applied to the innervated membrane of the isolated single cell of the Sachs electric organ (electroplax) of the electric eel, Electrophorus electricus. Concentrations as low as 0.05 μg/ml irreversibly antagonized depolarization by carbamylcholine, whereas concentrations of 0.1 mg/ml or higher were required to directly and irreversibly depolarize and block electrical excitation. The active component of the venom was stable to boiling at acid pH, destroyed by boiling at alkaline pH and nondialyzable and corresponded to those fractions containing maximum phospholipase A activity demonstrable when isolated by paper electrophoresis and Sephadex filtration. Phospholipase C and lysolecithin in concentrations of 1 mg/ml and 0.2 mg/ml, respectively, depolarized and blocked electrical excitation, whereas lower concentrations did not antagonize depolarization by carbamylcholine. Triton X-100 (0.01 mg/ml) antagonized carbamylcholine, whereas 10-fold higher concentrations directly blocked electrical excitation. Hyaluronidase had no effect on resting or action potential but decreased the depolarizing response to carbamylcholine. At minimal concentrations which blocked the depolarizing response to carbamylcholine, SV caused only slight splitting of phospholipids in single cells of the Sachs organ. A concentration (1 mg/ml) of SV which blocked electrical excitation caused 80–100 per cent splitting of lecithin, phosphatidylethanolamine and phosphatidylserine, the three principal phospholipids of the electric tissue. Similar percentages of splitting of the latter two phospholipids but only about one-third of the lecithin occurred at SV concentration of 0.1 mg/ml. These results indicate that electrical excitability in the eel electroplax can be maintained in the presence of extensive phospholipid splitting. Depolarization and block of electrical excitation by relatively high concentrations of SV may have resulted from splitting of phospholipids, especially lecithin, or may have reflected action of lysophosphatide detergents produced as a result of the action of phospholipase A upon membranal phospholipids.     

Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells

The steroid transformation of hydrocortisone to prednisolone, combining the two techniques of immobilized whole cells and high steroid concentrations, was investigated and found to be a feasible process. Freeze-dried Corynebacterium simplex cells were immobilized in collagen, tanned with glutaraldehyde, and cast into a membrane. The reaction was studied at hydrocortisone concentrations ranging from 5 to 50 mg/ml. The following aspects of the system were examined: (1) the substrate concentration effect upon the reaction; (2) the effect of enzyme concentration; (3) the rate-concentration relationship; and (4) the product inhibition characteristics of the system. The optimal substrate concentration was found to be 15 mg/ml of a membrane concentration of 80 mg/ml. This reaction attained an 80% conversion in 48 hr. A liner relation was found between the initial reaction rate and membrane concentration. One can thus increase the net production of steroid per unit volume and time by increasing the membrane levels. A physical limit to this increase occurred at membrane concentrations greater than 125 mg/ml. The rate-concentration relationship was linear when graphed on a Line weaver-Burk plot: giving a K_m′ and V_m′ value of 5.39 mg/ml and 0.556 mg/ml/hr, respectively. When the data were tested for competitive product inhibition, the curves fitted the experimental points fairly well and produced K_m′ and V_m′ values of 4.52 mg/ml and 0.566 mg/ml/hr, respectively. Product inhibition experiments showed that the inhibition was not purely competitive. At low substrate concentrations, product inhibited the enzyme; at high substrate concentrations, the enzyme was first stimulated and then depressed by increasing levels of products. This behavior has been analyzed and shown to be possibly a result of the information of a tertiary intermediate produced during the reaction.     

Parturition in beef cows following administration of porcine relaxin at ten days prepartum

Administration of procine relaxin (pRLX) to heifers 5 d prepartum has been reported to expedite parturition. Thirty-eight mature crossbred beef cows were randomly assigned to one of three treatment groups. Control animals (C; n = 13) received an intramuscular (i.m.) injection of 2 ml corn oil and 2 ml i.m. phosphate-buffered saline (PBS) 24 h later; relaxin treated animals (RLX; n = 13) received 2 ml i.m. corn oil and 1.0 mg i.m. pRLX 24 h later; estradiol-relaxin treated animals (E-RLX; n = 12) received 20 mg i.m. estradiol benzoate (EB) and 1.0 mg i.m. pRLX 24 h later. Treatment with pRLX occurred at 272.6+/-0.14 d of gestation. The pRLX had been purified to homogeneity from porcine ovaries collected during late pregnancy and was determined to have >/=3000 U/mg by the mouse interpubic ligament bioassay. Peripheral blood samples were collected from all cows at 0, 4, 8 and 24 h, respective to corn oil or EB administration, and assayed for plasma estradiol-17beta. At 24 h post administration of EB, plasma estradiol-17beta concentrations were 48.0+/-10.5 pg/ml for C and RLX cows and 178.5+/-14.8 pg/ml for E-RLX cows. There were no treatment effects (P>/=0.10) for elapsed time from treatment to parturition (304.2+/-22.4 h), gestation length (285.2+/-0.9 d), calving difficulty score (1.05+/-0.04), calf vigor score (1.05+/-0.04) or calf birth weight (38.0+/-0.88 kg). Additionally, there were no retained placental membranes in any cows. Administration of pRLX intramuscularly to beef cows at 10 d before expected parturition was not effective in inducing premature parturition. Furthermore, the effectiveness of pRLX in inducing parturition was not enhanced by pretreatment with estradiol benzoate.     

Toxic and mutagenic effects of carcinogens on the mitochondria of Saccharomyces cerevisiae.

Summary Nineteen haploid yeast (Saccharomyces cerevisiae) strains were used to assess the relative growth inhibitory potencies on fermentable vs. non-fermentable media of a collection of carcinogenic and noncarcinogenic chemicals. The majority of carcinogens were distinctly more potent on the non-fermentable (glycerol) medium, where mitochondrial function is required for growth, than on the fermentable medium, where it is not. The anti-mitochondrial selectivity indicated by these growth tests was much slighter for the non-carcinogens. Similarly most carcinogens induced the cytoplasmic petite mutation whereas the non-carcinogens did not.Five carcinogens which were tested impaired the development of cytochromes aa ₃ and b in glucose cultures.Six carcinogens, when tested, inhibited growth on three fermentable sugars, the utilisation of which requires mitochondrial function.Out of five carcinogens which were examined, four suppressed the surface-dependent phenomenon of flocculence in a flocculating strain of yeast, at concentrations primarily affecting the mitochondrial system; the fifth had a similar but less pronounced effect.     

PAF increases vascular permeability in selected tissues: effect of BN-52021 and L-655,240

The effect of the potent inflammatory mediator, platelet activating factor (PAF) was studied on the vascular permeability of selected rat tissues using the extravasation of Evans blue dye (EB) as a marker. EB (20 mg/kg) was injected in the caudal vein together with increasing doses of PAF (0.1, 1.0 and 5.0 micrograms/kg). The animals were killed and the dye was extracted in selected organs using formamide (4 ml/g wet weight tissues) and the content was expressed as EB micrograms/g dry weight. Extravasation of EB varied markedly from one tissue to another and increased as a function of time (from 0 to 60 min). PAF (5.0 micrograms/kg) increased the pancreas and duodenum vascular permeability by 15 and 5 fold respectively. At the doses of 0.1 and 1.0 microgram/kg, PAF induced a slight increase (P less than 0.01) of the vascular permeability of the heart 5 min after the injection. The PAF antagonist BN-52021 (2 and 10 mg/kg) produced a dose-dependent inhibition of the PAF effects on the pancreas, heart and duodenum. Maximum inhibition (approximately 100%) was achieved at the dose of 10 mg/kg. This antagonist given in the absence or the presence of PAF reduced the lung plasma extravasation below control levels. A thromboxane antagonist, L-655,240 (1.0 and 5.0 mg/kg) also inhibited PAF-induced increases in vascular permeability in heart, duodenum and pancreas. It also reduced below control levels the EB extravasation in kidneys, spleen and lungs. Maximum inhibition (50% for the duodenum, and 40% for the pancreas) was achieved at the dose of 5.0 mg/kg.     

Protective properties of butanolic extract of the Calendula officinalis L.(marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger

Abstract

Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O₂^?-) and hydroxyl radicals (HO^?) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC₅₀ values of 1.0 ± 0.09 mg/ml and 0.5 ± 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe²⁺/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC₅₀ = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in μM Trolox equivalents) was 368.14 ± 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 ± 14.5 μM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.     

Effects of inhibitors of integrin binding on cellular outgrowth from bovine inner cell masses in vitro

Summary Bovine inner cell masses (ICM) cultured on fibronectin give rise to extensive cellular outgrowths containing endoderm. Peptides with the Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Gly-Asp (RGD) sequences inhibit cell migration on fibronectin by binding to the fibronectin-recognition site in several integrins. To identify integrins involved in endodermal cell outgrowth on fibronectin and vitronectin, the effects of the EILDV and RGD peptides were evaluated in vitro. In experiment 1, ICM were cultured on fibronectin in medium containing 0.5 or 1.0 mg/ml EILDV or RGD (or both). Compared with 0 mg/ml, 0.5 mg/ml EILDV suppressed (P<0.10) outgrowth area overall, and 1.0 mg/ml EILDV reduced (P<0.05) outgrowth area after 72 h of culture. Compared with 0 mg/ml, 0.5 and 1.0 mg/ml RGD reduced (P<0.05) outgrowth area after 72 h of culture. Plasminogen activator activity in conditioned medium increased (P<0.05) in 0.5 mg/ml RGD but decreased (P<0.10) in 1.0 mg/ml RGD compared with 0 mg/ml RGD. In experiment 2, bovine ICM were cultured on vitronectin in medium containing 0.5 or 1.0 mg/ml RGD. Neither concentration of RGD (P>0.10) affected the extent of cellular outgrowth on vitronectin. Bovine endodermal cell migration on fibronectin can be modulated by the RGD and EILDV peptides. Despite inhibition, neither peptide completely prevented outgrowth on fibronectin. In contrast, cellular outgrowth on vitronectin was unaffected by RGD. The persistence of cellular outgrowth on fibronectin and the absence of inhibition by RGD for ICM cultured on vitronectin suggests that bovine endodermal cells can use alternative cellular adhesion systems, such as nonintegrin receptors, during outgrowth.