MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos.

The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro-produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico-predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro-produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P=0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development.

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation.

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

Isotope effects and intermediates in the reduction of NO by P450(NOR).

The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.

NIH shift in flavin-dependent monooxygenation: mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase.

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium Azoarcus evansii catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O2-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by 1H, 2H, and 13C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-2H]2-aminobenzoyl-CoA was converted into [6-2H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 --> 6 shift of the [5-2H] label. Label from NAD2H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D2O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1, 3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity.