L-tryptophan inhibits formation of mutagens during cooking of meat and in laboratory models

The formation of mutagens and carcinogens of the 2-amino-3-methylimidazo[4,5-f]quinoline and -quinoxaline type, formed during the frying or broiling of meats and in liquid-reflux laboratory models, was inhibited by L-tryptophan in a dose-dependent fashion. Addition of 75 mg (1.04 mg/cm2 surface area) of L-tryptophan per side in a sauce to ground beef patties prior to cooking significantly blocked the formation of mutagens observed in control patties treated identically with sauce but without L-tryptophan. The sauce itself did not have a significant inhibitory effect. When a mixture of 35 mM glucose, 70 mM glycine, and 70 mM creatinine in diethylene glycol-water (95:5) was heated in a liquid-reflux model for 2 h at 150 degrees C, the addition of 1.75-105 mM L-tryptophan gave a dose-related inhibition of mutagen formation, that reached 100% inhibition with 105 mM L-tryptophan.     

The mechanism of killing of mouse fibroblasts by the amino acid analogue 5-fluorotryptophan

The mechanism of killing of A9 fibroblasts by 5-fluorotryptophan has been studied. L-tryptophan competitively relieves the growth inhibition caused by 5FT. After incubation with 5FT, 3H-5FT was incorporated into protein, replacing tryptophan residues. During the initial hours of incubation with 5FT, a specific inhibition was observed of the incorporation of L-tryptophan into protein; later this inhibition was followed by a general inhibition of protein synthesis and cell division. However, nuclear division continued after cell division had ceased. While 5FT was observed to be incorporated into protein after a 1 hour period in MEM + 0.40 mM 5FT in A9, no 3H-5FT was incorporated into protein in a mutant isolated by its resistance to killingy by 5FT. These results support the hypothesis that cell death occurs due to malfunctioning proteins which contain 5FT residues.     

Use of bacterial gamma-glutamyltranspeptidase for enzymatic synthesis of gamma-D-glutamyl compounds

An enzymatic method for synthesizing various gamma-D-glutamyl compounds efficiently and stereospecifically involving bacterial gamma-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a gamma-glutamyl donor was developed. With D-glutamine as a gamma-glutamyl donor instead of L-glutamine in gamma-glutamyltaurine synthesis, by-products such as gamma-glutamylglutamine and gamma-glutamyl-gamma-glutamyltaurine were not synthesized and the yield of gamma-glutamyltaurine dramatically increased from 25 to 71%. It was also shown that the purification could be simplified without these gamma-glutamyl by-products. The possibility of synthesizing various gamma-D-glutamyl compounds was also shown.     

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells

The presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation.     

DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals

2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC(50)) and cytotoxicity (LC(50)) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage--both strand breaks and oxidised purines--was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

Studies on avian erythrocyte metabolism. XVI. Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes

The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined. Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate. Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h. Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension. Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions. The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation). Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions. The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr. The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds.     

Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST 1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.     

De novo emergence of insulin-stimulated glucose uptake in human aortic endothelial cells incubated with high glucose

Elevated glucose concentrations have profound effects on cell function. We hypothesized that incubation of human aortic endothelial cells (HAEC) with high glucose increases insulin signaling and develops the appearance of insulin-stimulated glucose uptake by the cells. Compared with 5 mM glucose, incubation of HAEC with 30 mM glucose for up to 48 h increased in a time-dependent manner expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, and GLUT1 proteins. High glucose also increased the specific binding of (125)I-labeled insulin in HAEC accompanied by accelerated production of interleukin (IL)-6 and IL-8. Short-term stimulation by 50 microU/ml insulin did not activate [(14)C]glucose uptake by HAEC incubated in 5 mM glucose. However, an addition of insulin to high glucose-exposed endothelial cells led to a significant increase in [(14)C]glucose uptake in a glucose concentration- and time-dependent fashion, reaching a plateau at 48 h of incubation. Furthermore, incubation of HAEC with 30 mM glucose resulted in a new insulin-stimulated extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase phosphorylation and increased lipid peroxidation and production of reactive oxygen species. These studies show for the first time that high glucose increases expression of insulin receptors and downstream elements of the insulin-signaling pathway and transforms "insulin-resistant" aortic endothelial cells into "insulin-sensitive" tissue regarding glucose uptake.     

Evidence for a pronounced secretion of cyclic AMP by Tetrahymena

The unicellular eukaryote Tetrahymena pyriformis secretes significant amounts of cyclic AMP into its external medium. Cells transferred from growth medium into any of the following three different non-nutrient media: (a) 5 mM phosphate buffer containing 47 mM NaCl and 1 mM MgSO4, (b) 10 mM Tris, or (c) 1.3 mM Tris containing 1 mM citrate and 1 mM Ca(OH)2, released to the outside almost 60--80% of the total cyclic AMP produced during 2--5 h of incubation. Tris-citrate-Ca+2 medium was chosen for further experiments because of its minimal nonspecific interference in the cyclic AMP radioimmunoassay. The identity of the secreted material recognized as cyclic AMP by radioimmunoassay was confirmed by demonstrating its almost complete hydrolysis with commerical beef heart phosphodiesterase. Furthermore, the radioimmunoassay-active material in the concentrated medium co-chromatographed on paper with [3H]cyclic AMP, as judged by assay of the eluted material. After resuspending cells in Tris-citrate-Ca2+ medium, the extracellular concentration of cyclic AMP rose steadily over a 5-h period, reaching a level equvalent to approximately 35--50 pmol cyclic AMP/10(6) cells vs. an internal cyclic AMP quantity at 5 h of 8--10 pmol/10(6) cells. After 5 h, the level of extracellular cyclic AMP reached a plateau. There was no degradation or uptake of external cyclic AMP by the cells during this period.     

Dissimilation of Tryptophan and Related Indolic Compounds by Ruminal Microorganisms In Vitro

Intraruminal doses of L-tryptophan cause acute pulmonary edema and emphysema in cattle. The D and L isomers of tryptophan and 22 related indolic compounds were incubated with ruminal microorganisms in vitro. Incubation of L-[U-benzene ring-(14)C]tryptophan with ruminal microorganisms for 24 h resulted in 39% of the added radioactivity being incorporated into skatole, 7% into indole, and 4% into indoleacetate (IAA). D-Tryptophan was not degraded to any of these metabolites. The major pathway of skatole formation from L-tryptophan appeared to be by the decarboxylation of IAA. Incubation of [2-(14)C]IAA with ruminal microorganisms for 24 h resulted in 38% incorporation into skatole. L-[5-Hydroxy]tryptophan was degraded to 5-hydroxyskatole and 5-hydroxyindole, whereas 5-hydroxyindoleacetate was degraded to only 5-hydroxyskatole. Incubation of indolepyruvate, indolelactate, and indolealdehyde with ruminal microorganisms resulted in the formation of both skatole and indole. Under similar conditions, indoleacetaldehyde was converted to IAA and tryptophol. The addition of increasing concentrations of glucose (0 to 110 mM) reduced the formation of both skatole and indole from L-tryptophan and resulted in the accumulation of IAA. Antibiotics reduced the degradation of L-tryptophan to skatole and indole, with kanamycin and neomycin particularly effective in reducing the decarboxylation of IAA to skatole.     

Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan

We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.     

Effect of protein kinase C activation on Na+-H+ exchange in erythrocytes of frog Rana temporaria

The treatment of frog erythrocytes incubated in standard nitrate medium with 100 nM phorbol ester (PMA) induced a sharp increase in the 22Na uptake by the cells and intracellular Na(+) concentration. The PMA-induced enhancement in 22Na uptake was stimulated by the addition of 0.1 mM ouabain to the incubation medium and completely blocked by 1 mM amiloride. The time course of 22Na uptake by frog red cells in the presence of PMA showed a lag phase ( approximately 5 min), after which was linear within 5-15 min. The calculated Na(+) influx in erythrocytes treated with PMA was 49.4+/-3.7 mmol l(-1) cells h(-1) as compared with 1.2+/-0.25 mmol l(-1) h(-1) for control cells. 5-(N-ethyl-N-isopropyl)-amiloride, selective blocker of NHE1, caused a dose-dependent inhibition of the PMA-induced Na(+) influx with IC(50) of 0.27 microM. The PMA-induced Na(+) influx was almost completely inhibited by 0.1 microM staurosporine, protein kinase C blocker. Pretreatment of frog red blood cells for 5, 10 or 15 min with 10 mM NaF, non-selective inhibitor of protein phosphatase, led to a progressive stimulation of the PMA effect on Na(+) influx. Both amiloride and NaF did not affect the basal Na(+) influx in frog erythrocytes. The data indicate that the Na(+)-H(+) exchanger in the frog erythrocytes is quiescent under basal conditions and can be markedly stimulated by PMA.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.     

Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. strain NGK1 in polyurethane foam

A Pseudomonas sp. strain NGKI (NCIM 5120) capable of degrading naphthalene was immobilized in polyurethane foam. The naphthalene-degrading activity of the freely suspended cells was compared with that of immobilized cells in batches in shaken culture and in a continuous culture system in a packed-bed reactor. Increasing concentrations of naphthalene were better tolerated and more quickly degraded by immobilized cell cultures than by free cells. An initial naphthalene concentration of 25 mM was completely degraded by freely suspended cells (4 x 10(10) cfu ml(-1)) and polyurethane-foam-immobilized cells (0.8-1 x 10(12) cfu g(-1) foam cubes) after 4 days and 2 days of incubation, respectively. Free cells degraded a maximum of 30 mM naphthalene after 4 days of incubation with 50 mM naphthalene, and no further degradation was observed even after 15 days of incubation, whereas foam-immobilized cells brought about the complete degradation of 50 mM initial naphthalene after 6 days of incubation. Furthermore, with 25 mM naphthalene, the polyurethane-foam-immobilized cells were re-used 45 times over a period of 90 days without losing naphthalene-degrading activity. By contrast, with the same amount of naphthalene, alginate-, agar-, and polyacrylamide-entrapped cells could be reused for 18, 12, and 23 times over a period of 44, 28, and 50 days, respectively. During continuous degradation in a packed-bed reactor, foam-immobilized cells degraded 80 mM naphthalene at a rate of 150 ml(-1) h(-1). With the same flow rate and 40 mM naphthalene, this system operated efficiently and continuously for about 120 days, whereas the packed-bed reactor with alginate-, agar-, and polyacrylamide-entrapped cells could be operated only for 45, 40, and 60 days respectively. Thus, more efficient degradation of naphthalene could be achieved by immobilizing cells of Pseudomonas sp. strain NGK1 in polyurethane foam, rather than in the other matrices tested.     

Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

Cell-free stimulation of the insulin-sensitive cAMP phosphodiesterase by the joint actions of ATP and the soluble fraction from insulin-treated rat liver

The insulin-sensitive cAMP phosphodiesterase (PDE) from rat adipocytes was stimulated 60-70% upon incubation with 2 mM ATP and the soluble fraction (Fraction S-1) from insulin-treated rat liver. The effect of ATP was partially mimicked by ATP-gamma-S or GTP, but not by AMP-PNP. The PDE-stimulating activity in Fraction S-1 was preserved in the presence of 50 mM sodium phenyl phosphate, 50 mM sodium fluoride, and 0.1 mM sodium vanadate. The PDE-stimulating activity was not inhibited with either 0.5 mM H-7 or 5 microM PKI-(5-24)-peptide, but was blocked with 1 mM Kemptide. The active component in Fraction S-1 may be a phosphorylated compound, which, in the presence of ATP, may mediate the hormonal action on PDE.