Effect of L-glutamine for freezing equine embryos: evaluation by DAPI staining and transfer of multiple embryos to recipient mares.

Day 6.5 equine embryos (n=30) were frozen in a medium containing glycerol (2.5-10.0%) supplemented with 0, 20 or 100 mmol L-glutamine 1(-1). After thawing, the embryos were tested individually, using 4',6'-diamidino-2-phenylindole (DAPI) staining to evaluate cell death. Three embryos (one frozen at each L-glutamine concentration) were transferred together into individual recipient mares. Pregnancy diagnosis was performed at day 12 (age of embryo). Embryos were collected at day 14 (age of embryo) and were identified by PCR amplified microsatellite analysis. Nine of ten recipient mares that received multiple embryos were pregnant: one mare was pregnant with two embryos and the other eight mares were pregnant with one embryo. The pregnancy rate was significantly higher (P < 0.05) for embryos transferred to recipient mares after freezing in media containing 100 mmol L-glutamine l(-1) (6 of 10 pregnancies) than for embryos frozen in media containing either 20 or 0 mmol L-glutamine l(-1) (1 of 10 and 3 of 10 pregnancies, respectively). Furthermore, DAPI staining analysis indicated that the percentage fluorescence was significantly lower (P < 0.05) in embryos frozen in media containing 100 mmol L-glutamine l(-1) (4.6 +/- 2.4, n=10) than in embryos frozen in media containing either 20 or 0 mmol L-glutamine l(-1) (23.0 +/- 31.3 and 10.8 +/- 9.5, n=10, respectively).

Triple helix formation on plasmid DNA determined by a size-exclusion chromatographic method.

Triple-helix-forming oligodeoxynucleotides are receiving considerable attention due to their potential applications for the inhibition of specific genes in vivo. However, their development is impaired by the lack of triple helix formation under physiological conditions. It is thus crucial to be able to quantitatively assay triple helix formation of various oligodeoxynucleotides on different target sequences. Usual methods to detect triple helix formation are restricted under the experimental conditions that can be studied. In addition, quantitative techniques are limited. We present a novel method for rapid detection and quantification of triple helix formation between an oligodeoxynucleotide and a plasmid carrying a target sequence. The oligodeoxynucleotide was radiolabeled and, after incubation with the target plasmid, the unbound oligodeoxynucleotide was separated from the mixture of plasmids and plasmid-bound oligodeoxynucleotides by rapid gel filtration spun columns. The formation of a triple helix between a target plasmid and several oligodeoxynucleotides was demonstrated and compared. Temperature, sequence and ionic dependencies, and kinetics of association were analyzed. This new technique can be used under a variety of conditions and should allow the rapid determination of optimal conditions required for triple helix formation, as well as the easy selection of an oligodeoxynucleotide that specifically binds with the highest affinity to a target double-stranded sequence.

Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography.

Plasmid DNA used for nonviral therapeutic gene transfer or nucleic acid vaccination has to be highly purified devoid of contaminating components such as bacterial proteins, endotoxins, or bacterial chromosomal DNA. We have developed a new affinity chromatography technique for plasmid DNA purification: triple-helix affinity chromatography (THAC). This technique is based on the sequence-specific interaction of an oligonucleotide forming a triple-helix with plasmid DNA. The oligonucleotide was covalently linked to a chromatographic matrix, thus providing a reusable affinity support. By inserting a suitable homopurine sequence in the plasmid DNA, it is possible to obtain a triple-helix interaction that will only be stable at mild acidic pH and that will dissociate in alkaline conditions. A crude lysate from a recombinant E. coli, or a pre-purified plasmid DNA, is thus applied at acidic pH on to a THAC column. After extensive washing of the column, purified plasmid DNA is eluted using an alkaline buffer. The binding conditions of the plasmid DNA on to the column have been optimized, as well as the hybridization sequence and the linker group between the matrix and the third strand oligonucleotide. The THAC technique makes it possible to purify in one step supercoiled plasmid DNA, and to significantly reduce the level of contaminating RNA, endotoxins and chromosomal DNA. In particular, a 100-fold reduction of chromosomal DNA contamination over that obtained with conventional techniques can be achieved through a single additional THAC step. Further improvements of THAC technology are possible, and we anticipate that this technique can be scaled up for integration into a full commercial-scale DNA production process.

Parentage testing of Day 10 equine embryos by amplified PCR analysis of microsatellites.

Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared to those of the dam to identify those of maternal origin. The paternal alleles were then searched for within the genotype of the 3 sires, and the stallion(s) that exhibited the particular allele was said to be compatible with the embryo for this microsatellite. In this way, the true sire was identified correctly for all 21 embryos.

Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining.

Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezing/thawing all the embryos were stained with DAPI. The percentage of dead cell area was significantly lower in the cooling groups than in the freezing groups and no significant differences were apparent between the cryoprotectants used in the study.

The effect of sucrose in the thawing solution on the morphology and mobility of frozen equine embryos.

Seventy-five embryos were collected 6 days after ovulation. Sixty embryos were frozen in straws using glycerol as the cryoprotectant in an automatic freezer. In Experiment 1 the freezing and thawing media were supplemented with 1.3 g/l PVP; in Experiment 2 the supplement was 5% FCS. The embryos were thawed for 30 s at +37 degrees C in a waterbath. In Experiment 1 glycerol was removed from 10 embryos in 6 steps. In 10 other embryos, glycerol and sucrose were both removed from the medium in 6 steps. After glycerol and sucrose removal, the embryos were stained with 4',6'-diamidino-2-phenylindole (DAPI) to count the percentage of dead cells. Fluorescent rate (FR) was defined as a ratio of fluorescent area versus total area. Mean (+/- s.d.) FR in this experiment was significantly lower (P<0.01) in embryos thawed with sucrose (0.28 +/- 0.13) than in embryos thawed with glycerol alone (0.53 +/- 0.25). In Experiment 2, 40 embryos were frozen and glycerol, with or without sucrose, was removed after thawing as for Experiment 1. Ten embryos in both groups were stained with DAPI. All the frozen-thawed embryos were transferred nonsurgically to recipient mares. Fourteen fresh embryos were transferred as controls, 7 of which were stained with DAPI before transfer. There was no difference in pregnancy rates between DAPI-stained versus nonstained embryos, indicating that the staining process had no negative effects on embryonic survival. Insufficient embryos were transferred to be able to demonstrate any difference in pregnancy rates between embryos thawed with or without sucrose in the medium.

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.

Reflex and direct effects of sodium cyanide in anesthetized rats.

Small doses of NaCN (250 micrograms.kg-1) intravenously injected into normal anesthetized Wistar rats, provoked immediate hyperventilation, bradycardia and systemic hypotension. These effects resulted mostly from peripheral chemoreceptor (PCR) stimulation. In totally baro- and chemodenervated rats (CDN), the drug induced hypoventilation, reduction in O2 consumption, prolonged bradycardia and systemic hypotension, characteristics of direct intoxication. In hyperoxic rats, hyperventilation was reduced. Bradycardia was suppressed with reduction in the systemic hypotension. These modifications arose partly from a reduction in PCR excitability and partly from a direct O2 activity counteracting cyanide poisoning. After chemical sympathetic denervation, rats presented prolonged and enlarged hyperventilation, bradycardia and systemic hypotension due to loss of adaptive baroreflex responses. Cyanide effects are predominantly reflexes in intact rats but these reactions mask the direct toxic effect of the drug which cannot be totally neglected.

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones.

A 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase-like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV subunits and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP-dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature-sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co-expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single-point mutations occurring in the 'quinolone resistance-determining region' (QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser-83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone-resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low-quinolone-resistant isolates. In contrast, Ser-80 of GrlA, which corresponds to Ser-83 of E. coli GyrA, is substituted to Phe or Tyr in both high- and low-quinolone-resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

Ventilatory responses to brief hypoxic stimuli after simulated altitude exposure in rat.

A blunted ventilatory response to acute hypoxia is described in altitude acclimatized small mammals. The peripheral or central origin of this blunting remains controversial. As the O2 sensitivity is always tested by steady-state steps of progressive hypoxia, an enhanced central inhibiting mechanism similar to those created by sustained hypoxia is suspected to be involved in the blunting. To avoid such central effect, we analyzed the ventilatory responses to very short glomic stimuli such as N2 tests and administration of NaCN by intravenous bolus in chronically hypoxic rats. The blunted ventilatory response was present only for hypoxemic hypoxia. A D2-receptor blockade failed to restore this response to those of littermate controls. We concluded that in rats, after altitude acclimatization, the central transduction of glomic afferences was intact because of the unchanged ventilatory response to cyanide. Reduced glomic sensitivity to low PO2 appeared specifically responsible for the blunted response to N2 tests with a very limited contribution from inhibitory dopaminergic mechanisms.

[Bilateral carotid occlusion in the rat neonatally treated with capsaicin]. / Occlusion bilatérale des carotides primitives chez le rat traité par capsicine à la naissance.

Bilateral carotid occlusion (BCO) was performed in pentobarbital anesthetized adult rats neonatally treated with capsaicin (50 mg/kg, sc, CNT rats). Pressor and ventilatory responses to BCO in CNT rats were compared with those of littermate controls injected with a same volume of solvent (olive oil, 0.1 ml). Capsaicin was used in order to produce partial degeneration of unmyelinated C fibres related to baroreflexes and peripheral chemoreflexes. In control rats, BCO provoked in less than 5 s, hyperventilation, hypocapnia and hyperoxia. Systemic arterial hypertension and tachycardia developed more slowly. They were maximum at 65 s. At this time, ventilation was returned to control values. Hyperventilation results from the stimulation of the carotid chemoreceptors by stagnant asphyxia generated by the blood flow stop. Hypertension and tachycardia are provoked by an increase in the orthosympathetic outflow when carotid baroreceptors are unloaded. In a first time, chemoreceptors stimulation tends to oppose to the increase of heart rate in normal rats. In a second one, development of hypertension is autolimited by the stimulation of the aortic baroreceptors particularly effective in rats. Simultaneously the hyperoxic inhibition from aortic chemoreceptors, the central hypocapnia and the reperfusion of the carotid bodies lead to the suppression of hyperventilation. As hyperventilation decreases when hypertension develops, even in rats with vago-sympathetic section at low cervical level, the part of aortic baroreceptors effects is probably reduced except for the fibres travelling through superior laryngeal nerves. Carotid bodies reperfusion seems to predominate. Before any manipulations, CNT rats had lower heart rate and systemic blood pressure than controls. During BCO, initial hyperventilation was moderately prolonged as hypertension slowly developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of morphological modifications of day 6.5 horse embryos after cryopreservation: differential effects on inner cell mass and trophoblast cells.

Sixteen embryos were recovered nonsurgically at day 6.5 after induced ovulation from Welsh pony mares and were evaluated for cellular changes that occur because of exposure to the cryoprotectant with or without the freeze and thaw process. Day 6.5 horse embryos were either (i) frozen and thawed using glycerol as cryoprotectant (n = 6), (ii) given only the glycerol treatment (n = 5), or (iii) washed in phosphate-buffered saline (PBS) the same number of times as in the glycerol treatment (n = 5). After treatments, embryos were incubated in Minimum Essential Medium (MEM), supplemented with BSA, glutamine, antibiotics and buffered with Hepes, for 1 h for one embryo per group and for 6 h for the others. After histological fixation, embryos were serially sectioned. On observation by light microscopy, the total numbers of interphasic, mitotic and pycnotic nuclei of each embryo were counted. Electron microscopy was used to evaluate the damage to the fine structure of intracellular organelles. The proportion of mitotic cells did not differ among groups (control: 2.3%; glycerol-treated: 1.8%; frozen-thawed: 1.3%). There were significant differences in the proportion of pycnotic cells both between control (12.8% +/- 5.6) and glycerol-treated embryos (39.4% +/- 15.9) (P < 0.05) and between control and frozen-thawed embryos (42.2% +/- 14.9) (P < 0.001), but no difference was found between treated embryos (glycerol-treated and frozen-thawed embryos). Degenerated cells were not localized in the same place in each embryo and no ultrastructural alteration was uniformly observed among every embryo of each group, but inner cell mass (ICM) cells were affected most by treatments (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation between the effects of zymosan on the systemic and pulmonary vessels of the rat.

1. Zymosan, an activator of the alternative complement pathway, (2 to 16 mg kg-1) injected intravenously via the tail vein of anaesthetized rats, dose-dependently increased the vascular permeability of lung parenchyma, as measured by the accumulation of 125I-labelled albumin in lungs. 2. Pretreatment of the animals with cyclo-oxygenase inhibitors, indomethacin or ketoprofen (3 mg kg-1) or with the lipoxygenase and cyclo-oxygenase inhibitor, BW755C (40 mg kg-1) abolished the vascular permeability changes induced by zymosan (16 mg kg-1). Neither, the PAF antagonist, WEB 2086 (10 mg kg-1) nor the antagonist of mast cell amines, mepyramine and methysergide (3 mg kg-1) affected the plasma exudation in lungs. Zymosan did not induce any accumulation of labelled albumin in lungs of rats made leukopenic by rabbit anti-neutrophil serum. 3. Zymosan (16 mg kg-1) increased the haematocrit. This increase was not modified by indomethacin but reduced by WEB 2086. 3. Intravenous injection of zymosan (3 and 8 mg kg-1) in anaesthetized rats transiently increased right ventricular blood pressure and pulmonary arterial pressure, accelerated respiratory rate and decreased systemic blood pressure. 5. WEB 2086 largely reduced the systemic hypotension but did not affect the increase of pulmonary vascular resistance. Indomethacin inhibited the increase of blood pressure in the right ventricle and the modification of the respiratory rate. This drug did not inhibit but increased the systemic hypotension induced by zymosan. 6. Zymosan (16 mg kg-1) reduced serum complement haemolytic activity by 46%. 7. These data suggest that the pulmonary vascular changes induced by intravascular complement activation with zymosan in rats are mediated by neutrophils and prostanoids while the systemic vascular effects depend mainly on PAF.

Interference of lung distension with the cardiovascular response to chemoreceptor stimulation in anaesthetized rats.

Experiments were performed to examine the role of the stimulation of pulmonary stretch receptors in cardiovascular response to peripheral chemoreceptor stimulation in spontaneously breathing anaesthetized rats. The effects of continuous positive tracheal pressure (0.1 to 0.4 kPa) were examined in normal rats and in rats pretreated with almitrine bismesylate, a potent stimulator of the arterial chemoreceptors. In the untreated group, an increase of a maximum 4 +/- 0.7 beats.min-1 in heart rate was found during testing at pressure of 0.2 and 0.3 kPa when interference with augmented breath was avoided. These pressures at the tracheal level had no effect on systemic blood pressure, so that baroreflex influences can be discounted. During strong almitrine-induced chemostimulation, a prolonged bradycardia developed with the long-lasting hyperventilation. The same continuous positive pressures were unable to overcome the chemoreflex bradycardia. No changes in heart rate were observed under these conditions, whatever the potentiation of the mecanoreceptors for distension in the presence of alveolar hypocapnia. It was concluded that the stretching of lung mecanoreceptors on the cardiac control is of little importance in anaesthetized rats and that bradycardia generally dominates during strong stimulation of the chemoreceptors even when lung distension is artificially increased.

[Chemostimulation and catecholamine content of the rat adrenal medulla]. / Chémostimulation et teneur de la médullo-surrénale du rat en catécholamines.

Unanaesthetized rats whose arterial chemoreceptors were stimulated by an one hour acute exposition to hypoxic gaseous mixtures with various carbon dioxide concentrations, presented depletion of the catecholamines content of their adrenal glands only when hypocapnia or increased pH was present (non compensated hypoxia). Moreover, exposition to simultaneous hypoxia and hypercapnia increased the epinephrine stock of the adrenal glands. No changes were found in the myocardium amine content in the same conditions. When anaesthetized rats were treated by iv injection of almitrine bismesylate, a peripheral chemoreceptors stimulating drug, adrenal catecholamines content was insignificantly reduced. In the myocardium, the amines remained at control levels. The most powerful factor related to catecholamines depletion in the adrenals seems to be the hypocapnia or the alkalosis induced by the hyperventilation provoked by glomic stimulation. No indication has been found in favor of an effective adrenergic stimulation caused directly by chemoreceptors stimulation.

[Pathogenic mechanisms of acute pulmonary edema of hemodynamic origin in rats]. / Mécanismes pathogéniques de l'oedème pulmonaire aigu d'origine hémodynamique chez le rat.

In normal anaesthetized rats (pentobarbital, 40 mg/kg i.p.), intravenous injection of a bolus of vasopressin (0.3 micrograms/kg) provoked a large increase in pulmonary and in systemic blood pressures. About three minutes later, some rats (60%) developed an acute pulmonary edema (OPA), froth appearing at the trachea. Other animals presented no OPA at the 4th minute following the injection, but OPA appeared immediately when bilateral vagotomy was performed at that time. Factors explaining the appearance of OPA are mechanical ones, circulatory or respiratory, without interferences with autonomous nervous processes.

The effect of cryopreservation on the metabolic activity of day-6.5 horse embryos.

The decrease in embryo viability caused by cryopreservation may be due, in part, to metabolic disturbances. To determine the effect of cryopreservation on metabolism, Day -6.5 horse embryos were either frozen and thawed using glycerol as the cryoprotectant, given only the glycerol treatment or washed an equal number of times in phosphate buffered saline (PBS). Before and after treatment, individual embryos were incubated with L-[14C(U)]-glutamine, to measure Krebs cycle activity, and D-[5-3H]-glucose, to measure Embden-Meyerhof pathway activity. Before treatment, glucose metabolism ranged from 110-625 pmol/2 h and glutamine metabolism from 4.1-15.9 pmol/2 h, both being highly correlated with embryo volume. Mean glucose metabolism in the control group increased 76% between the pre-treatment and post treatment measurements compared with 1% in the pooled treated groups, whereas mean glutamine metabolism increased only 10% in the control group but 50% in the treated embryos. Before treatment, there was no difference in mean ratio of glucose to glutamine metabolism between groups, but after treatment this ratio was almost 2-fold greater in the control group than in the treated group. These results indicate that cryopreservation inhibits anaerobic glucose metabolism and stimulates aerobic glutamine metabolism. However, this is an effect of the cryoprotectant, rather than of freezing and thawing.