Mitochondrial Genetics. VI the Petite Mutation in SACCHAROMYCES CEREVISIAE: Interrelations between the Loss of the ρ+ Factor and the Loss of the Drug Resistance Mitochondrial Genetic Markers

The survival of the ρ⁺ factor and of Drug^R mitochondrial genetic markers after exposure to ethidium bromide has been studied. A technique allowing the determination of Drug^R genetic markers among a great number of both grande and petite colonies has been developed. The results have been analyzed by the target theory. The survival of the ρ⁺ factor is always less than the survival of any Drug^R genetic marker. The survivals of C^R and E^R are similar to each other, while that of O^R is greater than that of the other two Drug^R markers. All possible combinations of Drug^R markers have been found among the ρ^- petite cells induced, while the only type found among the grande colonies is the preexisting one. The loss of the C^R and E^R genetic markers was found to be the most frequently concomitant, while the correlation between the loss of the O^R marker and the other two Drug^R markers is less strong. Similar results have been obtained after U.V. irradiation. Interpretations concerning the structure of the yeast mitochondrial genome are given and hypotheses on the mechanism of petite mutation discussed.     

The autonomic transfer of mitochondrial factors (cytoduction) during crossing of Saccharomyces cerevisiae cells]

When crossing the genetically marked yeast strains obtained from the Gif collection we observed the appearance of haploid nucleo-cytoplasmic hybrids carrying the 3 nuclear markers of the rho- parent and the mitochondrial markers (rho+ ER CR) of the other parent. The frequency of such cytoduction was about 1 per cent. The mitochondrial markers ER and CR were transmited to cytoductants together and did not segregate. The possible mechanisms of the cytoduction and its significance are discussed.     

Extrachromosomal inheritance in Schizosaccharomyces pombe

Summary Primary and secondary spore clones were analyzed from two- and three-factor crosses involving the mitochondrial markers conferring resistance to antimycin (A ^R ), chloramphenicol (C ^R ), and erythromycin (E ^R ). As in zygote clones (Seitz-Mayr et al., 1978), transmission of markers is higher in two-factor trans-crosses than in cis-crosses. Except transmission of C ^R in the cross A ^R C ^R E ^R xA ^S C ^{S E S} , no significant differences between cis- and trans-configuration were observed in three-factor crosses. In contrast to zygote clones, in spore clones transmission rates of the two or three markers in a given cross are roughly equal. 18 out of 20 secondary spore clones of different mitochondrial phenotypes appeared to be homoplasmic, whereas 2 still continued to segregate. One of these spore isolates was analyzed, and segregation was found to continue for more than 150 generations after spore germination. Whereas up to more than 80% of zygote clones in certain crosses were uniform, only 2 out of 91 tetrads were uniform, i.e. all four spores were homoplasmic for the same mitochondrial genotype. Presence or absence of recombinant mitochondrial phenytypes among secondary spore clones from tetrads indicated, whether, cytoplasmic mixing had occurred in the original zygote or not. Within an ascus, the number of spores containing recombinant genotype(s) is a direct measure for the extent of cytoplasmic mixing in the zygote. In 82 tetrads analyzed, the number of tetrads with 0, 1, 2, 3, and 4 spores containing recombinant genotype(s) were 25, 37, 14, 5, and 1, respectively. In conclusion, the extent of cytoplasmic mixing at the cell stage before forespore membrane formation is highly variable.     

Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. II. DNA-DNA hybridization studies and buoyant density determinations

Buoyant density of mitochondrial DNA from 14 cytoplasmic petite mutants issued from the same grande yeast Saccharomyces cerevisiae was determined. Mutants that have retained the mitochondrial gene conferring resistance to erythromycin displayed higher buoyant density, while mutants that have retained the mitochondrial gene conferring resistance to chloramphenicol displayed lower buoyant density. It is inferred that the segment which carries the E^R gene has a higher G + C content than the segment which carries the C^R gene. DNA-DNA filter hybridizations were carried out systematically in different reciprocal pair-wise combinations between mtDNAs purified from various mutants and from the grande. All petites were found to be deleted in 42 to 93% of the grande sequence, depending on the mutant studied. Sequence homology between petite mtDNAs was greatest in mutants retaining common genetic markers and was least when different genetic markers were retained. Practically no hybridization was found between some C^RE^O and C^OE^R mutants. Correlations established between the extent of DNA-DNA hybridization, kinetic and genetic complexity show that a selective enrichment of gene specific sequences occurs in mtDNA of petites.     

A structured model for the kinetics of fungal amylase production

Summary The cell is considered to be divided into nucleic acids, proteinaceous material and storage compounds. The enzyme is believed to be constitutive but repressed by the rate of catabolism. A structured model is developed to describe the growth, amylase production and dissolved oxygen profile in the batch culture ofAspergillus oryzae.Nomenclature CA Conc. of enzyme SKB units/m³ - C_D,C_E,C_G,C_O,C_S Conc. of D.E.G. mass, oxygen, substrate kg/m³ - C_O* Mean oxygen conc. in gas-liquid interface kg/m³ - C_X Total cell conc. kg/m³ - D D-mass (proteins) kg - E E-mass (storage carbon) kg - G G-mass (necleic acids) kg - K_E Rate of usage of E-mass kg/m³/h - K_EO K_E with oxygen limitation kg/m³/h - Q Active fraction of promotor genes - - R_D,R_E,R_G Rate of production of D,E,G-mass kg/m³/h - R_O,R_S Rate of usage of oxygen, substrate kg/m³/h - R_DO,R_EO,R_GO R_D,R_E and R_G with oxygen limitation kg/m³/h - S Substrate (carbon) concentration kg/m³ - t Time h - t_o Time at which C_S = 0 h - K₁,K₂,K₃,K₂₄,K₂₅ Stoichiometric constants - - K₄,K₅,K₆,K₇ Rate constants h^–1 - K₈-K₁₂,K₁₈,K₂₀-K₂₃ Michaelis-Menten constants kg/m³ - K₂₆,K₂₆ Absorption coefficient, K₂₆ at C_X = 0 h^–1 - K₂₇ Empirical constant kg/m³ - K₁₅ Rate of enzyme formation SKB units/kg - K₁₆,K₁₇ Equilibrium constants m³/kg - K₁₉ Decay constant for mRNA h^–1     

Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

Summary A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the ^+– mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of ^– cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.Abbreviations E.B. Ethidium bromide - E.M.S. Ethyl méthane sulfonate - C^S/C^R Allelic forms of the rib 1 locus conferring chloramphenicol sensitivity/resistance - E^S/E^R Allelic forms of the rib 3 locus conferring erythromycine sensitivity/resistance - O^R/O^R Allelic forms of the oli 1 locus conferring oligomycin sensitivity/resistance - P^S/P^R Allelic forms of the par 1 locus conferring paromomycine sensitivity/resistance - D^S/D^R Allelic forms of the diu 1 or diu 2 loci conferring diuron sensitivity/resistance - C^S/C^R Allelic forms of the mitochondrial locus - ⁺ grande or respiratory competent cells - ^– petite or cytoplasmic respiratory deficient cells     

Cytoduction as a new tool in studying the cytoplasmic heredity in yeast

Summary When crossing the haploid cells of genetically marked yeast strains we observed the appearance of both normal diploid zygotes and haploid nuclear cytoplasmic hybrids. The latter had the nuclear markers of one and the cytoplasmic marker (rho⁺) of the other parent. The autonomous cytoplasmic factor transfer was termed as cytoduction. Cytoduction is supposed to be the abortive form of yeast cell mating. Only about 1% of cytoductants is usually observed.Cytoduction can be used as a simple test on cytoplasmic determination of some characters. We observed the transfer into cytoductant cells of not only rho⁺ marker but of resistance factors to antibiotics (erythromycin, neomycin) and killer factor as well. Cytoduction can be applied towards constructing strains having the identical nucleus genotype with mitochondria and other cytoplasmic factors of different origin.In crossing strains with doubly marked mitochondria recombination of mitochondrial markers in cytoductant haploid cells was observed, the pattern of which was similar to that of mitochondrial recombination in normal zygotes.     

Isolation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in extracts from human plasma

All-E-(3R,3′R,6′R)-lutein, all-E-(3R,3′R)-zeaxanthin, all-E-(3R,3′S,6′R)-3′-epilutein and some geometrical isomers of the former two dihydroxycarotenoids have been separated from an extract of human plasma by semipreparative high-performance liquid chromatography on a silica-based nitrile-bonded column. In the order of chromatographic elution, the isolated fractions were identified as all-E-lutein, all-E-zeaxanthin, all-E-3′-epilutein, 9Z-lutein, 9′Z-lutein, a mixture of 13Z-lutein and 13′Z-lutein, 9Z-zeaxanthin, 13Z-zeaxanthin and 15Z-zeaxanthin. The structures of all compounds, including the relative configuration at C(3′) and C(6′) of the luteins and the position of the stereomutated double bonds in the geometrical isomers, were unambiguously established by ¹H nuclear magnetic resonance spectroscopy. The absolute configuration of the three all-E compounds was derived by circular dichroism and is also assumed to be valid for the geometrical isomers. The ultraviolet—visible absorption and mass spectra of each of the individually isolated compounds were also in agreement with the proposed structures.     

Fusion of mitochondria with protoplasts in Saccharomyces cerevisiae.

Summary Protoplasts prepared from a neutral petite haploid BO60AF-1 (a ade2 arg4 leu2 trp C ^O E ^O O ^{O O O}) were mixed with mitochondria isolated from an oligomycin resistant respiring haploid AN^ROR 12D (a his4 leu2 thr4 C ^S E ^S O _II ^{R + +}) and treated with 30% polythylene glycol and CaCl₂. When the treated protoplasts were spread and incubated on selective agar plates, oligomycin resistant respiration-sufficient colonies appeared with low frequency. All of these colonies carried the mitochondrial genotype of C ^S E ^S O _II ^{R + +} and showed the same mating type and nutritional requirements as did BO60AF-1, thus evidencing the mitochondrial transfer into protoplasts. Recombination and transmission of the mitochondrial drug resistance markers were studied in crosses involving the strains issued from mitochondria accepted protoplasts.     

Carbon Isotope Composition of Nighttime Leaf-Respired CO2 in the Agricultural-Pastoral Zone of the Songnen Plain,Northeast China

Variations in the carbon isotope signature of leaf dark-respired CO₂ (δ¹³C_R) within a single night is a widely observed phenomenon. However, it is unclear whether there are plant functional type differences with regard to the amplitude of the nighttime variation in δ¹³C_R. These differences, if present, would be important for interpreting the short-term variations in the stable carbon signature of ecosystem respiration and the partitioning of carbon fluxes. To assess the plant functional type differences relating to the magnitude of the nighttime variation in δ¹³C_R and the respiratory apparent fractionation, we measured the δ¹³C_R, the leaf gas exchange, and the δ¹³C of the respiratory substrates of 22 species present in the agricultural-pastoral zone of the Songnen Plain, northeast China. The species studied were grouped into C₃ and C₄ plants, trees, grasses, and herbs. A significant nocturnal shift in δ¹³C_R was detected in 20 of the studied species, with the magnitude of the shift ranging from 1‰ to 5.8‰. The magnitude of the nighttime variation in δ¹³C_R was strongly correlated with the daytime cumulative carbon assimilation, which suggests that variation in δ¹³C_R were influenced, to some extent, by changes in the contribution of malate decarboxylation to total respiratory CO₂ flux. There were no differences in the magnitude of the nighttime variation in δ¹³C_R between the C₃ and C₄ plants, as well as among the woody plants, herbs and graminoids. Leaf respired CO₂ was enriched in ¹³C compared to biomass, soluble carbohydrates and lipids; however the magnitude of enrichment differed between 8 pm and 4 am, which were mainly caused by the changes in δ¹³C_R. We also detected the plant functional type differences in respiratory apparent fractionation relative to biomass at 4 am, which suggests that caution should be exercised when using the δ¹³C of bulk leaf material as a proxy for the δ¹³C of leaf-respired CO₂.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an α-fetoprotein-secreting hepatoma

The purpose of this work was to study the effect of α-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-17β (E₂)-dependent marker, i.e., progestophilins in E-sensitive cells C₂9RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of E₂, while they proliferate in culture regardless of the presence of E₂. C₂9RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3 × 10⁻¹⁰ M E₂ in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3 × 10⁻⁸ M E₂ in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 11-Methoxy-17β ethynylestradiol (R₂₈₅₈), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E₂ and with R₂₈₅₈.In addition, we compared the free E₂ levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E₂ concentrations above 11 pg E₂/ml, whereas there was no correlation between the free E₂ levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E₂ below 0.4 pg/ml, whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E₂ of 44 pg/ml. We conclude that the effect of hepatoma serum on the E₂ induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than E₂ trapping by AFP.     

Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Optimum temperature policy for batch reactors performing biochemical reactions in the presence of enzyme deactivation

A necessary condition is found for the optimum temperature policy which leads to the minimum reaction time for a given final conversion of substrate in a well stirred, enzymatic batch reactor performing an enzyme-catalyzed reaction following Michaelis-Menten kinetics in the presence of first order enzyme decay. The reasoning, which is based on Euler's classical approach to variational calculus, is relevant for the predesign steps because it indicates in a simple fashion which temperature program should be followed in order to obtain the maximum advantage of existing enzyme using the type of reactor usually elected by technologists in the fine biochemistry field. In order to highlight the relevance and applicability of the work reported here, the case of optimality under isothermal operating conditions is considered and a practical example is worked out.List of Symbols C _E mol.m^–3 concentration of active enzyme - C _E ^* dimensionless counterpart of C_E - C _E,0 mol.m^–3 initial concentration of active enzyme - C _E,b mol.m^–3 final concentration of active enzyme - C _E,opt ^* optimal dimensionless counterpart of C_E - C _smol.m^–3 concentration of substrate - C _S ^Emphasis>/* dimensionless counterpart of C_S - C _S,0mol.m^–3 initial concentration of substrate - C _S,bmol.m^–3 final concentration of substrate - E enzyme in active form - E ₃ ^* dimensionless counterpart of E_a,3 - E _a,1J.mol^–1 activation energy associated with k₁ - E _a,3J.mol^–1 activation energy associated with k₃ - E _d enzyme in deactivated form - ES enzyme/substrate complex - k ₁ s^–1 kinetic constant associated with the enzyme-catalyzed transformation of substrate - k _1,0 s^–1 preexponential factor associated with k₁ - k ₂ mol^–1.m³s^–1 kinetic constant associated with the binding of substrate to the enzyme - k _–2 s^–1 kinetic constant associated with the dissociation of the enzyme/substrate complex - K _2,0 mol.m^–3 constant value of K₂ - K _2,0 ^* dimensionless counterpart of K_2,0 - k ₃ s^–1 kinetic constant associated with the deactivation of enzyme - k _3,0 s^–1 preexponential factor associated with k₃ - k _3,0 ^* dimensionless counterpart of k_3,0 - P product - R J.K^–1.mol^–1 ideal gas constant - S substrate - t s time since start-up of reaction - T K absolute temperature - T ^* dimensionless absolute temperature - T _i,opt ^* optimal dimensionless isothermal temperature of operation - T _opt ^* optimal dimensionless temperature of operation - t _b s time of a batch - t _b ^* dimensionless counterpart of t_b - t _b,min ^* minimum value of the dimensionless counterpart of t_b Greek Symbols dimensionless counterpart of C_E,0 - dimensionless counterpart of C_E,b - dummmy variable of integration - dummy variable of integration - auxiliary dimensionless variable - ^* dimensionless variation of k₁ with temperature - _i ^* dimensionless value of k₁ under isothermal conditions - _opt ^* optimal dimensionless variation of k₁ with temperature     

Role of lipids in theNeurospora crassa membrane

Summary The effect of doubling the saturated fatty acid content on the electrophysiology ofNeurospora crassa membranes was studied. Intracellular membrane input resistance (R _m ) and potential (E _m ) were measured for wild-type (w/t) andcel ^– (Tween 40) organisms as a function of temperature. Over the 0 to 40°C temperature range studied, meanE _m values of bothw/t andcel ^– (Tw 40) organisms increased from –160 to –210 mV. This difference is greater than that expected from Nernst potential considerations, indicating an active component ofE _m . This active component is insensitive to a doubling of the saturated fatty acid content.R _m exhibits a temperature dependence and hysteresis. Averaged data indicate an increase inR _m with decreased temperature. The slope of the temperature dependence varies among individual hyphae. Above 17.5°Ccel ^– (Tw 40) hyphae averaged greater than 70% higher values ofR _m thanw/t. Below 17.5°Cw/t R _m data divided into low and high temperature dependence groups, whilecel ^– data exhibited a low temperature dependence. The results are discussed in relation to gel-liquid crystal phase transitions, membrane fluidity, and the contribution of fatty acid structure to membrane electrical properties.     

Biogenesis of Mitochondria: Analysis of Deletion of Mitochondrial Antibiotic Resistance Markers in Petite Mutants of Saccharomyces cerevisiae

Yeast strains carrying markers in several mitochondrial antibiotic resistance loci have been employed in a study of the retention and deletion of mitochondrial genes in cytoplasmic petite mutants. An assessment is made of the results in terms of the probable arrangement and linkage of mitochondrial genetic markers. The results are indicative of the retention of continuous stretches of the mitochondrial genome in most petite mutants, and it is therefore possible to propose a gene order based on co-retention of different markers. The order par, mik1, oli1 is suggested from the petite studies in the case of three markers not previously assigned an unambiguous order by analysis of mitochondrial gene recombination. The frequency of separation of markers by deletion in petites was of an order similar to that obtained by recombination in polar crosses, except in the case of the ery1 and cap1 loci, which were rarely separated in petite mutants. The deletion or retention of the locus determining polarity of recombination (ω) was also demonstrated and shown to coincide with deletion or retention of the ery1, cap1 region of the mitochondrial genome. Petites retaining this region, when crossed with rho⁺ strains, display features of polarity of recombination and transmission similar to the parent rho⁺ strain. By contrast a petite determined to have lost the ω⁺ locus did not show normal polarity of marker transmission. Differences were observed in the relative frequency of retention of markers in a number of strains and also when comparing petites derived spontaneously with those obtained after ultraviolet light mutagenesis. By contrast, a similar pattern of marker retention was seen when comparing spontaneous with ethidium bromide-induced petites.     

Changes in membrane potential, membrane resistance, and intracellular H, K, Na, and Cl activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H⁺, K⁺, Na⁺, and Cl⁻ ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential (E_m) and electrical resistance (R_m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R_m gradually increased. By the end of maturation of Pleurodeles oocytes E_m had stabilized at about −10 mV and R_m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E_m spontaneously shifted from about −15 to about +30 mV; simultaneously R_m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K⁺ activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na⁺ activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na⁺, accompanied by an increase in Cl⁻ activity, was observed as soon as E_m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E_m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E_m.     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 μM Resveratrol (C^R, C⁻ respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C⁻V^R, C^RV^R) or without (C^–V^-, C^RV⁻) 0.5 μM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in C^RV^R group compared with C^RV⁻ group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but C^RV⁻ and C^RV^R groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C⁻V^R) restored GSH content (P < 0.05) to the level found in the C^R group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 μM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.