Delivery rates after elective single cryopreserved embryo transfer related to embryo survival.

OBJECTIVE: The objective of this study was to assess if eSCET (elective Single Cryopreserved Embryo Transfer) outcome is related to blastomere survival rate. The final objective was to avoid multiple pregnancies and offer the best chances to women to achieve pregnancy even during their frozen-thawed embryo transfer (FET) cycles. STUDY DESIGN: Patients were included in this prospective observational study if they met the following criteria: (i) women age <37 years old; (ii) IVF of ICSI cycle rank ≤2, (iii) eSET proposed during fresh embryo transfer cycle and (iv) ≥1 good quality cryopreserved embryos available (<20% fragmentation and 4-5 blastomeres at day-2 or 7-9 blastomeres at day-3). Live birth rates (LBR) were compared into eSCET groups according to embryo survival (partially damaged or intact transferred embryo). RESULTS: We observed among selected patients, that partial loss of blastomeres (1 blastomere for day-2 embryos, 1 or 2 blastomeres for day-3 embryos) following FET cycles did not affect LBR compared with intact embryo. CONCLUSION: These results underline the relevance of eSCET as a strategy to reduce multiple pregnancies frequency without reducing LBR.

Phosphorylation of anti-HIV nucleoside analogs by nucleoside diphosphate kinase.

The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.

Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides.

The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.

Does a secular trend exist in the distribution of occlusal patterns?

The existence of a secular trend in the distribution of occlusal patterns was studied in two generations of children. Study models and demographic data of a sample of 265 children from the previous generation (group A) and recordings of clinical examinations of 988 children from the present generation (group B) served as the data base for this study. Children in whom caries affected the occlusion and those in the deciduous dentition stage were excluded. Thus, occlusal analysis was performed for 102 children in group A and 703 in group B. A dramatic decrease was found in the prevalence of caries affecting the occlusion. No difference existed between the two groups with respect to molar and canine anteroposterior relationships. However, there was a decrease in the prevalence of normal occlusion accompanied by an increase of Class I malocclusion.

X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase.

To be effective as antiviral agent, AZT (3'-azido-3'-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-A x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3'OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the beta- and gamma-phosphate, which plays an important part in phosphate transfer.

Phosphorylation of nucleoside diphosphate kinase at the active site studied by steady-state and time-resolved fluorescence.

Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.

Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum.

Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5' non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.