Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h^?1 L^?1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)^?1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.     

Stability of chromatophores during ATP regeneration in an enzyme reactor

ATP regeneration from ADP and inorganic phosphate catalysed by chromatophores from Rhodo-spirillum rubrum has been coupled to the production of glucose-6-phosphate from glucose catalysed by hexokinase. Different ADP derivatives were tested in respect of their function as substrates for the chromatophores, but only 3′NH₂-ADP was phosphorylated. Several factors causing inactivation of the chromatophores during operational conditions were studied. The addition of radical scavengers resulted in an increased stability of the chromatophores, and it was concluded that free radicals had been formed in the presence of oxygen and caused damage to the membranes. In the presence of catalase, a stable ATP production was obtained for 6 h at an activity of 4.5 mol ATP × (mol bacteriochlorophyll)⁻¹ min⁻¹. In a nitrogen flushed reaction medium, the optimum concentration of the redox buffering ascorbic acid changed during the course of several hours of illumination, while it did not change in an air atmosphere.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport.

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μ ) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μ -succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μ ; 4 μ -antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μ ) and antimycin A (4 μ ) by 110%. Azide (1 m ) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 m -KCN ( ). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca²⁺]_i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y₂ receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca²⁺-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y₂ receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Reduction of daunorubicin in the presence of sulfur-containing peptides

Daunorubicin, an anthracycline antitumor antibiotic, was reduced in the presence of reduced (GSH) or oxidized (GSSG) glutathione to evaluate the possibilities of detoxification or of potentiation of the drug by these compounds. The reductants were ^.COO⁻ free radicals produced by γ radiolysis. In both cases, the final product is 7-deoxydaunomycinone, i.e., the same as without glutathione. The reduction yield is also the same as without GSH or GSSG (0.23 μmol·J⁻¹). No glutathione depletion was observed. Limits for the rate constants of some possible nonenzymatic detoxification reactions are given. To evaluate the possible interactions of daunorubicin with sulfur-containing proteins, the reduction of this drug by ^.COO⁻ free radicals was also studied in the presence of a polypeptide containing two disulfide bridge are, respectively, 0.23 μmol·J⁻¹ 7-deoxydaunomycinone. The yields of reduction of the drug and of a protein disulfide bridge are, respectively, 0.23 μmol·J⁻¹ and ≤ 6 nmol·J⁻¹. These values indicate thet disulfide radical anions of the protein can reduce the drug, giving back the disulfide bridge, but that the drug transients niether oxidize nor reduce the protein.     

Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series

The effect of dilution rate on the production of lactic acid from whey permeate by Lactobacillus helveticus has been investigated. In the first chemostat of a two-stage system, total conversion (98.1%) and maximum lactic acid concentration (43.7 g l⁻¹) were obtained at a dilution rate (D_Itot) of 0.06 h⁻¹. Maximum volumetric productivities of lactic acid (8.27 g l⁻¹ h⁻¹) and biomass (1.90 g l⁻¹ h⁻¹) occurred at D_Itot of 0.40 h⁻¹. The fraction of -lactate in the product was found to increase with dilution rate and reached a maximum of 66% at the same dilution rate. The maximum specific growth rate (μ_max) on this medium was 0.7 h⁻¹. A Y_ATP (max) value of 22.4 g dry weight (mol ATP)⁻¹ and a maintenance coefficient of 8.0 mmol ATP (g dry weight h)⁻¹ were determined. The second stage, in series with the first, confirmed these results and further showed that the total residence time could be reduced by 50%, compared with a single chemostat for the same nearly complete level of substrate conversion.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.