Amburana cearensis leaf extract maintains survival and promotes in vitro development of ovine secondary follicles.

The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.

Effect of hypoxanthine, antioxidants and allopurinol on cholinesterase activities in rats.

In the present study, we investigate the in vitro effect of hypoxanthine on acetylcholinesterase and butyrylcholinesterase activities in the hippocampus, striatum, cerebral cortex and serum of 15-, 30- and 60-day-old rats. Furthermore, we also evaluated the influence of antioxidants, namely α-tocopherol (trolox) and ascorbic acid, and allopurinol to investigate the possible participation of free radicals and uric acid in the effects elicited by hypoxanthine on these parameters. Acetylcholinesterase and butyrylcholinesterase activities were determined according to Ellman et al. (Biochem Pharmacol 7:88-95, 1961), with some modifications. Hypoxanthine (10.0 µM), when added to the incubation medium, enhanced acetylcholinesterase activity in the hippocampus and striatum of 15- and 30-day-old rats and reduced butyrylcholinesterase activity in the serum of 60-day-old rats. The administration of allopurinol and/or antioxidants partially prevented the alterations caused by hypoxanthine in acetylcholinesterase and butyrylcholinesterase activities in the cerebrum and serum of rats. Data indicate that hypoxanthine alters cholinesterase activities, probably through free radicals and uric acid production since the alterations were prevented by the administration of allopurinol and antioxidants. It is presumed that the cholinesterase system may be associated, at least in part, with the neuronal dysfunction observed in patients affected by Lesch-Nyhan disease. In addition, although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins and allopurinol might serve as an adjuvant therapy to avoid progression of brain damage in patients affected by this disease.

Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.

DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative.

Effects of folate cycle disruption by the green tea polyphenol epigallocatechin-3-gallate.

We demonstrate that the tea polyphenol, epigallocatechin-3-gallate, is an efficient inhibitor of human dihydrofolate reductase. Like other antifolate compounds, epigallocatechin-3-gallate acts by disturbing folic acid metabolism in cells, causing the inhibition of DNA and RNA synthesis and altering DNA methylation. Epigallocatechin-3-gallate was seen to inhibit the growth of a human colon carcinoma cell line in a concentration and time dependent manner. Rescue experiments using leucovorin and hypoxanthine-thymine medium were the first indication that epigallocatechin-3-gallate could disturb the folate metabolism within cells. Epigallocatechin-3-gallate increased the uptake of [(3)H]-thymidine and showed synergy with 5-fluorouracil, while its inhibitory action was strengthened after treatment with hypoxanthine, which indicates that epigallocatechin-3-gallate decreases the cellular production of nucleotides, thus, disturbing DNA and RNA synthesis. In addition to its effects on nucleotide biosynthesis, antifolate treatment has been linked to a decrease in cellular methylation. Here, we observed that epigallocatechin-3-gallate altered the p16 methylation pattern from methylated to unmethylated as a result of folic acid deprivation. Finally, we demonstrate that epigallocatechin-3-gallate causes adenosine to be released from the cells because it disrupts the purine metabolism. By binding to its specific receptors, adenosine can modulate different signalling pathways. This proposed mechanism should help us to understand most of the molecular and cellular effects described for this tea polyphenol.

Antifolate pseudo-resistance due to elevated levels of thymidine and hypoxanthine in a commercial serum preparation.

BACKGROUND: Batch variability of sera used for cell culture is of considerable experimental concern. A novel fetal calf serum product, FCS Gold, was claimed to be the first defined fetal calf serum free of batch variation. MATERIALS AND METHODS: The efficacy of methotrexate (MTX) and LY231514 (multitargeted antifolate, MTA) in CCRF-CEM cells and KB cells was compared using media supplemented with FCS Gold or conventional fetal bovine serum. RESULTS: IC50 values from tests using conventional serum corresponded to published data. FCS Gold fully protected the cells from antifolate drug cytotoxicity. Dialysis of FCS Gold restored responsiveness to antifolate drugs. Elevated levels of hypoxanthine and thymidine were present in FCS Gold. They were approximately 10-fold greater than the concentrations required to overcome growth arrest mediated by 2 microM MTX. CONCLUSION: FCS Gold or identical products, e.g. FBS Gold, should not be used in studies on antifolate drug action.

Effects of culture media and energy sources on the inhibition of nuclear maturation in bovine oocytes.

The influence of the culture medium and energy sources on spontaneous nuclear maturation and inhibition of maturation in bovine cumulus-enclosed oocytes (CEO) was examined. CEO were cultured in Medium 199, minimum essential medium, M16, or synthetic oviduct fluid (SOF), all containing 3 mg/mL bovine serum albumin (BSA), and SOF without BSA, alone or supplemented with hypoxanthine (HYPO, 4 mM) or forskolin (FSK, 100 microM) for 21 h. More CEO remained at the GV stage in M16 compared to other media (P < 0.05). Supplementation with HYPO increased and FSK reduced the percentage of CEO remaining at the GV stage (P < 0.05) only in M16. The effects of energy sources, in the absence or presence of HYPO or FSK, were examined in CEO cultured in M16 salts+PVA. Glucose (0.5 and 5.5 mM), pyruvate (0.32 and 3.2 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of CEO remaining at the GV stage compared to M16 salts alone; only glutamine significantly increased the percentage of CEO at the MII stage compared to M16 salts. In M16 salts+HYPO, glucose (0.5 mM), pyruvate (0.32 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of GV and degenerate oocytes and increased the percentage of CEO at the MI stage. In M16 salts+FSK, the energy sources significantly decreased the percentage of oocytes with condensed chromosomes and increased the percentage of CEO reaching metaphase I. In conclusion, meiotic inhibitors had different effects in different culture media and glucose, pyruvate, lactate and glutamine were stimulatory to nuclear maturation. It was noteworthy that some of the results obtained were contrary to previous findings in mouse oocytes.

Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

Nitric oxide influences the maturation of cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and hypoxanthine-supplemented medium through different signaling pathways.

Nitric oxide (NO) has been recently shown to act with a dual action in mouse oocyte meiotic maturation depending on its concentration, but the mechanism(s) through which it influences oocyte maturation has not been fully clarified to date. The purpose of this study was to test the hypothesis that different signaling mechanisms exist for NO-stimulated and NO-inhibited in vitro maturation of meiosis in cumulus cell-enclosed oocytes (CEOs) from PMSG-primed immature female mice. CEOs were cultured in both spontaneous maturation model and hypoxanthine (HX) arrested model to investigate the mechanism(s). Sodium nitroprusside (SNP, an NO donor) at a concentration of 1mM delayed significantly germinal vesical breakdown (GVBD) during the first 5 h of incubation period and further inhibited the formation of first polar body (PB1) at the end of 24 h of incubation. While SNP, at a concentration of 10 microM, stimulated significantly the meiotic maturation of oocytes by overcoming the inhibition of HX. Methinine blue (MB, 10 microM) or 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 microM)), two soluble guanylate cyclase (sGC) inhibitors, could reverse SNP-inhibited spontaneous oocyte maturation, but had no effect on SNP-stimulated meiotic maturation in the presence of HX. 8-Br-cGMP (1mM), a cell-permeating cGMP analogue, demonstrated a significant inhibitory effect on both spontaneous meiotic maturation and HX-arrested meiotic maturation. The delay effect of SNP on GVBD occurrence was similar to that of forskolin (6 microM, an adenylate cyclase stimulator) and rolipram (250 microM, a phosphodiesterase 4 inhibitor), two cAMP elevating reagents. Both forskolin and rolipram reversed significantly the SNP-stimulated meiotic maturation, but did not reverse the SNP-inhibited spontaneous meiotic maturation. Cilostamide (1 microM), the selective inhibitor of phosphodiestrase 3 (PDE3), could mimic the inhibitory effect of HX on the spontaneous meiotic maturation in CEOs and this inhibitory effect could also be reversed by SNP (10 microM). Moreover, sphingosine (3 microM), a protein kinase C (PKC) inhibitor, blocked the SNP-inhibited spontaneous meiotic maturation, but did not block the SNP-stimulated meiotic maturation. Clearly, these results suggest that pathway differences are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation of mouse oocytes.

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water.

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.

Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro.

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro-L-arginine methyl ester (L-NAME) or N(w)-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and L-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10(-3) M L-NAME (P<0.01) or 10(-3) M L-NNA (P<0.01) and the inhibition could be reversed by the addition of SNP (10(-5) M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10(-7), 10(-6), 10(-5) M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1-4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM L-arg and 1 mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.

Allantoin, the oxidation product of uric acid is present in chicken and turkey plasma.

Urate oxidase is not present in birds yet allantoin, a product of this enzyme, has been measured in birds. Studies were designed to compare the concentrations of plasma purine derivatives in chickens and turkeys fed inosine-supplemented diets. The first study consisted of 12 male chicks that were fed diets supplemented with 0.6 mol inosine or hypoxanthine per kilogram diet from 3- to 6-week-old. Study 2 consisted of 12 turkey poults (toms) fed inosine-supplemented diets (0.7 mol/kg) from 6- to 8-week-old. Plasma allantoin and oxypurines concentrations were measured weekly using high performance liquid chromatography. Plasma uric acid (PUA) in chickens fed inosine-supplemented diets increased from 0.31 to 1.34 mM (P<0.05) at the end of week 2. In turkeys, those fed control diet had 0.17 mM PUA concentration compared to 0.3 mM in those fed the inosine diet at week 2 (P<0.05). Allantoin concentration increased in chickens from week 1 to 2 while a decrease was observed in turkeys (P<0.005) for both treatments. These data show that allantoin is present in turkey and chicken plasma. The presence of allantoin in avian plasma is consistent with uric acid acting as an antioxidant in these species.

Protection of insulin-secreting INS-1 cells against oxidative stress through adenoviral-mediated glutathione peroxidase overexpression.

OBJECTIVES: A large fraction of an islet graft can be lost early following allotransplantation from various non specific mechanisms including oxidative stress. Overexpression of antioxidant enzymes could confer a beneficial effect on islets exposed to reactive oxygen and nitrogen species. We examined the viability of beta cells driven to overexpress glutathione peroxidase (GPx) and exposed to a superoxide donor (hypoxanthine/xanthine oxidase HX/XO) and a nitric oxide donor (3-morpholinosydnonimine SIN-1). METHODS: Cultured INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying GPx cDNA (AdGPx). Additional experiments were performed with an adenovector carrying Cu/Zn superoxide dismutase cDNA (AdSOD). Cellular viability was tested by the WST-1 colorimetric assay and functionality by static incubation. RESULTS: AdGPx increased GPx activity within 48 hours from 0 (untransfected cells) to 60 +/- 11 U/g (cells transfected at an MOI of 25: 1). GPx overexpression significantly reduced cytotoxicity induced by HX/XO from 10.81 +/- 1.41 to 5.42 +/- 2.62% at 10 mU/ml and from 61.19 +/- 4.17 to 52.9 +/- 4.39% at 20 mU/ml (p=0.0002, transfected cells vs control cells). Doses of SIN-1 from 600 to 1000 micromol/l resulted in cytotoxicity ranging from 17.66 +/- 3.48 to 45.97 +/- 6.48% in control cells and from 5.65 +/- 1.37 to 35.80 +/- 5.59% in AdGPx transfected cells (p=0.015). The combination of AdGPx and AdSOD did not exhibit any synergistic cytoprotective effect. Control cells exposed to a HX/XO stress exhibited a reduction in glucose-theophylline stimulated insulin secretion by half, while stressed GPx overexpressing-cells maintained the same insulin secretion level than non-stressed cells. CONCLUSIONS: Adenoviral-induced overexpression of GPx enhances the resistance of a rat beta cell line to both reactive oxygen (ROS) and reactive nitrogen species (RNS) cytotoxicity. Transposition of these findings to human islet transplantation with a clinically-relevant procedure deserves further investigations.

Mammalian fibroblasts lacking mitochondrial NAD+-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase are glycine auxotrophs.

Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3-10 x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.

Osteosarcoma cells, resistant to methotrexate due to nucleoside and nucleobase salvage, are sensitive to nucleoside analogs.

PURPOSE: To test a novel strategy for overcoming intrinsic resistance to methotrexate (MTX) in osteosarcoma (OS) due to nucleoside and nucleobase salvage (NS). METHODS: Four OS cell lines, found to be highly resistant to MTX, were tested to determine the dominant mechanism of resistance. Sensitivity to MTX was tested in the presence of dialyzed serum or the transport inhibitor dipyridamole (DP) to confirm the contribution of NS to MTX resistance. We then investigated whether increased NS activity could be exploited using cytotoxic nucleoside analogs. RESULTS: Like other cell types, OS cells are capable of circumventing inhibition of de novo nucleotide synthesis by relying on NS. MTX, at concentrations as high as 1 m M did not inhibit cell growth in culture medium supplemented with undialyzed serum. In contrast, when NS was inhibited by DP or in medium depleted of nucleosides and nucleobases, sensitivity to MTX was seen at nanomolar concentrations. In medium with dialyzed serum, thymidine and hypoxanthine provided dose-dependent protection from MTX toxicity at concentrations similar to those seen in human plasma. No evidence of other significant mechanisms of resistance were found. All four cell lines were sensitive to 3-day exposures to cytarabine (IC50 0.22 to 2.88 micro M) and vidarabine (IC50 0.09 to 0.95 micro M). CONCLUSIONS: Salvage of de novo nucleotide synthesis inhibition by extracellular thymidine and hypoxanthine, at physiologically relevant concentrations, contributes to resistance to MTX in OS. However, this same process may impart a collateral sensitivity to nucleoside analogs. These findings support clinical trials for patients with OS using nucleoside analogs, either alone or in combination.

Antiplasmodial activity of aspidosperma indole alkaloids.

The antiplasmodial activity of twelve alkaloids with an aspidospermane skeleton was estimated in vitro on chloroquine-resistant and sensitive strains of Plasmodium falciparum. Seven tetracyclic alkaloids possessing a free ethyl chain such aspidospermine, showed IC50 after incubation for 72 h between 3.2 and 15.4 microM. Moreover, four pentacyclic alkaloids with ethyl chain included in a tetrahydrofuran, such haplocine, showed a reduced activity, with IC50, after 72 h, between 22.6 and 52.6 microM. According to these results, a chloroquine-potentiating experiment was also performed with two of the most active compounds. Isobolograms were obtained and demonstrated a synergic effect of N-formyl-aspidospermidine and aspidospermine when associated with chloroquine. The cytotoxicity and the selectivity index of some alkaloids were also estimated.

Manipulation of plasma uric acid in broiler chicks and its effect on leukocyte oxidative activity.

Birds have high metabolic rates, body temperatures, and plasma glucose concentrations yet physiologically age at a rate slower than comparably sized mammals. These studies were designed to test the hypothesis that the antioxidant uric acid protects birds against oxidative stress. Mixed sex broiler chicks (3 wk old) were fed diets supplemented or not with purines (0.6 mol hypoxanthine or inosine). Study 1 consisted of 18 female Cobb x Cobb broilers that were fed purines for 7 days, whereas study 2 consisted of 12 males in a 21-day trial. Study 3 involved 30 mixed sex broilers that were fed 40 or 50 mg allopurinol/kg body mass (BM) for 21 days, a drug that lowers plasma uric acid (PUA). PUA and leukocyte oxidative activity (LOA) were determined weekly for all studies. For study 2, pectoralis major shear force, relative kidney and liver sizes (RKS and RLS), and plasma glucose concentrations were also determined. In study 1, PUA concentration was increased three- and twofold (P < 0.001) in birds fed inosine or hypoxanthine, respectively, compared with control birds. LOA of birds supplemented with inosine was lower (P < 0.05) than that of control or hypoxanthine birds. In study 2, PUA concentrations were increased fivefold (P < 0.001) in birds fed inosine and twofold (P < 0.001) in birds fed hypoxanthine compared with control birds at day 21. RKS (g/kg BM) was greater (P < 0.001) for chicks fed purine diets compared with control chicks. Muscle shear value was lower (P < 0.05) in chicks fed purine diets. PUA concentration was decreased (P < 0.001) in birds consuming allopurinol diets, whereas LOA was increased (P < 0.01) in study 3. These studies show that PUA concentrations can be related to oxidative stress in birds, which can be linked to tissue aging.

Protein kinase C, rather than protein kinase A is involved in follicle-stimulating hormone-mediated meiotic resumption of mouse cumulus cell-enclosed oocytes in hypoxanthine-supplemented medium.

It has been reported that protein kinase C (PKC) activation participated in the porcine and bovine oocyte maturation, but not in mouse oocyte maturation in vitro. In the present study, the activators and inhibitors of protein kinase A (PKA) (forskolin, CDPKI and MDL-12230A) or PKC (PMA, staurosporine and sphingosine) were used to investigate the in vitro effect of PKA or PKC on spontaneous murine oocyte maturation, oocyte resumption of meiosis from HX inhibiting medium (medium+HX), and follicle stimulating hormone (FSH)-induced oocyte maturation. The results showed that when cumulus cell enclosed oocytes (CEOs) or denuded oocytes (DOs) were cultured for 24 h in the medium supplemented with forskolin (5 microM), an activator of adenylate cyclase, the spontaneous oocyte maturation were inhibited. A transient exposure (2 h) to forskolin (2-10 microM) in the medium+HX, and then transferred to a new medium+HX for the further culture, stimulated CEO resumption of meiosis. CDPKI (10(-10)-10(-6) M), an inhibitor of PKA, also stimulated oocyte meiotic maturation of CEO in the medium+HX, but not on DO. However, MDL-12230A (10(-12)-10(-9) M), an inhibitor of adenylate cyclase, did not promote oocyte maturation in HX arrested CEO. CDPKI (10(-10)-10(-6) M) or MDL-12230A (10(-12)-10(-9) M) had no effect on FSH-stimulated oocyte meiotic resumption, except at high doses of CDPKI (10(-7)-10(-6) M) or MDL-12230A (10(-9) M) which inhibited the FSH-induced formation of the first polar body (PB1). An activator of PKC, PMA (10(-11)-10(-7) M) dose-dependently inhibited spontaneous oocyte maturation of CEO or DO. Inhibitors of PKC, staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) induced oocytes in CEOs to resume meiosis in the presence of HX in a dose dependent manner, but had no effect on DOs. FSH (50IU/L) stimulated mouse oocytes in CEOs to override the arrest of HX and resume meiosis, while PMA, at the level of 10(-8)-10(-6) M, dramatically inhibited the stimulatory effect of FSH. These results indicate that PKC or PKA may be implicated in the regulation of mouse oocyte maturation. Thus while sustained high level of cAMP or PKA inhibit the resumption of meiosis, a transient rise in cAMP or PKA levels promotes oocyte maturation. The activation of PKC can also block oocyte meiotic resumption. Thus the inactivation of PKC, instead of the transient rise of PKA activity, appears to be involved in the process of FSH-mediated oocyte meiotic maturation.

Antiplasmodial and antioxidant isofuranonaphthoquinones from the roots of Bulbine capitata.

The roots of B. capitata yielded the new compounds 5,8-dihydroxy-1-tigloylmethylnaphtho[2,3-c]furan-4,9-dione, 1-acetoxymethyl-8-hydroxynaphtho[2,3-c]furan-4,9-dione, and 1-acetoxymethyl-5,8-dihydroxynaphtho[2,3-c]furan-4,9-dione, in addition to the known compounds chrysophanol, 10,10'-chrysophanol bianthrone, 8-hydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione, 5,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione, 5,8-dihydroxy-1-hydroxymethylnaphtho[2,3-c]furan-4,9-dione, and 8-hydroxy-5-methoxy-1-methylnaphtho[2,3-c]furan-4,9-dione, or 5-hydroxy-8-methoxy-1-methylnaphtho[2,3-c]furan-4,9-dione. The new as well as the known isofuranonaphthoquinones showed antioxidant and weak antiplasmodial activities.

In vitro culture of bovine preantral follicles.

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture. The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them. From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.

In vitro methylation of arsenite by rabbit liver cytosol: effect of metal ions, metal chelating agents, methyltransferase inhibitors and uremic toxins.

The methylation of carrier-free 74As-arsenite by liver cytosol of Flemish Giant rabbits is highly susceptible to additions of trace elements. In vitro supplementation of essential trace elements like zinc (Zn2+), vanadium (V5+), iron (Fe2+), copper (Cu2+) and selenate was shown to increase the methylation efficiency. Trivalent metal ions (e.g. Al3+, Cr3+ and Fe3+), Hg2+, Tl+ and SeO3(2-) had a deleterious effect. The inhibitory effect of EDTA, oxime and many divalent cations (Ca2+, Mg2+, Sr2+, ...) suggest a co-factor role for a specific divalent metal ion, possibly Zn2+. Chelating agents used in clinical treatment of acute and chronic inorganic arsenic poisoning lower the methylation capacity of cytosol by rendering the trivalent arsenic unavailable for the methyltransferase enzymes. S-adenosylhomocysteine and periodate-oxidized adenosine, inhibitors of s-adenosylmethionine dependent methylation pathways, inhibit the methylation of arsenite. Pyrogallol, a catechol-O-methyltransferase inhibitor, blocks the action of arsenite- and monomethylarsonic methyltransferase enzymes, suggesting a close structural relationship between the active sites of the different enzymes. Some uraemic toxins, namely oxalate, p-cresol, hypoxanthine, homocysteine and myo-inositol, inhibit arsenic methylation.