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  • Here the so called alkali


    Here the so-called alkali assay [18] is proposed as a method to be applied directly on the purified recombinant enzyme sharing the same structural and functional features of the wild-type enzyme [19], [20], [21] using its physiological substrate androstenedione. The assay that was already successfully applied on the cytochrome P450 BM3 for the screening of libraries of substrates [18], [22], is based on the detection of the so-called alkali product that forms upon strong alkali treatment of the NADP+ released during enzyme turnover. More specifically, in the presence of NaOH the NADP+ is firstly converted into an unstable pseudobase, a para-substituted NADP+ derivative that absorbs at 347nm and decays in 1.5h. Upon condensation of the pyridinium and the ribose rings of the pyridinic coenzyme a stable product, the alkali product, is formed, absorbing at 360nm and exhibiting a maximum fluorescence emission at 455nm [23], [24].
    Materials and methods
    Results and discussion
    Conclusions The results show that the method can be used as a technique to be applied in a microtiter plate format for the rapid fluorometric detection of NADP+ released during enzyme turnover. Despite the alkali assay is an indirect measurement of the aromatase activity, it is a cheap method that can be applied to other NAD(P)H dependent enzymes that are potential molecular targets of EDCs, including the other cytochromes P450 involved in steroidogenesis. Moreover, the assay uses the physiological substrate androstenedione that exhibits KM values reported to be in the nM-low μM range [21], [41], difficult to reach by non-natural substrates that have been already used for fluorometric assays to detect aromatase inhibition in high throughput formats. The assay represents a first test to identify potentially toxic compounds since their endocrine disrupting activity should then be confirmed by in vivo experiments. It has also to be taken into account that aromatase-inhibiting EDCs can act by altering aromatase expression due to the lack of the negative feedback exerted by estrogens through the hypothalamus-pituitary-gonadal axis [42]. For this reason, the assay should be combined with other techniques detecting possible EDCs-induced changes in aromatase levels.
    Transparency document
    Background The demand for treatments used to control the estrous TP-0903 in cattle may be illustrated by estimates of the use of artificial insemination (AI) and embryo transfer (ET). A conservative estimate of the worldwide use of AI is 83 million cows per year – representing about 20% of the breedable cattle population [1]. In addition, approximately 1 million embryos involving approximately 200,000 donors and 1 million recipients are produced world-wide each year by in vivo and vitro fertilization [2]. Assuming that synchronization treatments are used for only 10% of cows that are artificially inseminated (i.e., 2% of world population) and 50% of donors and recipients used for embryo transfer (conservative numbers), a total of 8.9 million synchronization treatments are given annually. More recent data from Brazil shows that the use of AI has increased sharply in the last 6 years to more than 10 million cows per year. More than 50% are now done by fixed-time AI (FTAI); that is, >6 million cows are synchronized per year in Brazil alone [3]. Further, the use of synchronization treatments is expected to expand with the current increase of in vitro embryo production in cattle where numbers have risen 100-fold over the last 10 years [2]. The identification of prostaglandin F2α (PGF) as the luteolysin responsible for regression of the corpus luteum (CL) in cattle provided a new means for controlling the length of the luteal phase and ovulation (reviewed in Ref. [4]). Several protocols involving different doses and intervals between doses of prostaglandins have since been designed [5], [6], but the effectiveness of PGF-based synchronization is limited by two things: 1) The growing CL is refractory to PGF during the first 5–6 days after estrus [5], and 2) PGF does not influence the state of follicular readiness to respond to an LH surge. The state of maturity of the dominant follicle at the time of PGF-induced luteolysis will determine the interval to estrus and ovulation which ranges from 1 to 6 days [5], [6]. Two doses of PGF 11–14 days apart is a common practice on many dairy and beef farms based on the rationale that approximately 67% of the animals (those with a CL ≥ 5 day-old or those experiencing natural luteal regression) should respond to the first PGF treatment and 100% should have a functional, PGF-responsive CL when the second dose is administered. The use of luteolytic doses of prostaglandin, however, still relies on estrus detection efficiency to provide acceptable outcomes. Herd heat detection rates range from 30% to 65% in high producing dairy farms [7], [8] and between 50% and 70% in commercial beef farms [7], [9]. Consequently, the rate of submission of animals for AI after detected estrus limits the effectiveness of the PGF protocol.