Peut-on optimiser la congélation des spermatozoïdes testiculaires? L’expérience du Centre Hospitalier de Poissy Saint-Germain

50% or more of non-obstructive azoospermic men have no spermatozoa in their testicular tissue, and no non-invasive predictor of spermatogenesis is yet available. For this reason, we therefore performed all TESE (74 TESE for non-obstructive azoospermia and 37 TESE for obstructive azoospermia) prior to initiating ovarian stimulation. 34% (25/74) of TESE performed for non-obstructive azoospermia were successful. Spermatozoa were retrieved in 100% of cases of obstructive azoospermia. When TESE were positive, spermatozoa were frozen in 25–50 μl micro-droplets (several straws). 60 ICSI cycles (25 couples) were treated for non obstructive azoospermia. The clinical pregnancy rate per ICSI cycle was 18%, and the implantation rate per embryo transferred was 9.2%. 81 ICSI cycles (37 couples) were treated for obstructive azoospermia. The fertilization rate was 54%, and embryo transfer was performed in 89% (72/81) of cycles. The clinical pregnancy rate per embryo ICSI cycle was 26%, and the implantation rate per embryo transferred was 16%. This management of azoospermic patients, including TESE and multiple testicular tissue freezing in micro-droplets prior to ovarian stimulation, avoids ova pick-up cancellation and multiple TESE, as several ICSI can be performed after a single TESE. Our results show that this micro-technique for freezing testicular tissue is effective not only for obstructive azoospermia, but also for non-obstructive azoospermia when only very few spermatozoa can be extracted from the testis.     

Synthesis and characterization of immobilized dopamine beta-hydroxylase in membrane-bound and solubilized formats

Dopamine beta-hydroxylase (DBH) catalyzes the beta-hydroxylation of dopamine to norepinephrine. The enzyme in chromaffin granules occurs in a soluble form and a form confined to the surrounding membrane. DBH was noncovalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase and the resulting DBH-IAM stationary phase was enzymatically active and was shown to mimic the membrane-bound form of the enzyme. DBH was also covalently immobilized onto a silica-based support containing, glutaraldehyde-P (Glut-P). The resulting DBH-Glut-P interphase was also enzymatically active, reproducible and shown to display characteristics of the solubilized enzyme. The results demonstrate that the different immobilization methods utilized for the enzyme can be used to quantitatively and qualitatively determine the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions for the two distinct forms of the enzyme. These new entities can be used in basic biochemical studies as well as in high throughput screening of substances for DBH substrate/inhibitor properties.     

Pharmacokinetics of (R)- and (S)-cyclophosphamide and their dechloroethylated metabolites in cancer patients

The complete pharmacokinetics (PK) of (R)- and (S)-cyclophosphamide (CP) and their dechloroethylated (DCE) metabolites have not been reported to date. We collected plasma and urine samples from 12 cancer patients and determined concentrations of both enantiomers of CP and DCE-CP using a chiral GC-MS method. All concentrations of (R)-CP, (S)-CP, (R)-DCE-CP, and (S)-DCE-CP were simultaneously modeled using an enantiospecific compartmental PK model. A population PK analysis was performed. Enantiospecific differences between (R)- and (S)-CP were found for the formation clearance of CP to the DCE metabolites (Clf: 0.25 (R) vs. 0.14 (S) L/h). No difference was found between enantiomers for Cl40H, Cld, Cl(m)R, ClT, or T1/2. In contrast to the adolescent and adult group of patients, a child (6 years old) appeared to have a very different PK and metabolic profile (Bayesian control analysis). Proportions of the (R,S)-CP doses transformed to the (R)-DCE- and (S)-DCE-CP were much higher (R: 25 vs. 1.9%, and S: 38 vs. 3.6%), while formation of active metabolites was much lower (R: 42 vs. 74%, and S: 48 vs. 77%). CP appears to be enantioselectively metabolized to the DCE metabolites. This PK model can evaluate the proportion of a CP dose that is transformed to toxic or active metabolites. It may therefore be used to optimize CP treatment, to identify important drug interactions and/or patients with an abnormal metabolic profile.     

Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.     

Evidence of renal metabolism of ifosfamide to nephrotoxic metabolites

Nephrotoxicity is a limiting factor in the use of ifosfamide in children. Despite the co-administration of uroprotective agents such as sodium 2-mercaptoethanesulfonate (mesna), ifosfamide chemotherapy is associated with nephropathy characterized by glomerular toxicity and Fanconi syndrome in many children treated with this drug. This is in distinction to cyclophosphamide, an analogue which differs solely by the position of a chloroethyl group, and which is not associated with nephrotoxicity. We hypothesized that ifosfamide is metabolized by cytochrome P450 (CYP) enzymes located in the renal tubular cell to the toxic metabolite chloroacetaldehyde; and, that the higher production of chloroacetaldehyde from ifosfamide than from cyclophosphamide explains the clinical differences in nephrotoxicity. We found that in both pig renal cortical microsomes and whole human kidney microsomes incubated with 1 mM ifosfamide for 3 hr, 2 and 3 dechloroethylifosfamide (DCEI) were produced. Our study provides evidence that porcine and human kidney microsomes are capable of biotransforming ifosfamide to DCEI metabolites that are produced in equimolar amounts with chloroacetaldehyde, indicating that chloroacetaldehyde is locally produced by renal cells as a possible mechanism for nephrotoxicity.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases α and δ) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication. © 1996 Wiley-Liss, Inc.     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.