Materials and Methods Recombinant protein, reagents and chemicals
Purified LTA4 hydrolase was a gift from Dr. Agnes Rinaldo Mattis, Karolinska Institute. Presequence peptidase was a gift from Prof. Elzbieta Glaser, Stockholm University. Mca-R-P-P-G-F-S-AF-K(Dnp)-OH was from R&D Systems, UK. Alanine-4-nitroanlide and other standard chemicals were from Sigma-Aldrich, Sweden.Simulation of progress curves
To simulate reaction progress curves expressing product concentration as a function of time, the reversible MM rate law for initial reaction velocity (Equations S1, equation 2) was numerically integrated according to Euler’s method with an integration time step of 1 second (Equations S1, equation 3). A function to estimate enzyme half life was also included as a mechanism-free model that accounts for enzyme inactivation by reducing its concentration with respect to its half-life (Equations S1, equation 4). Because product inhibition and the reversed reaction may be prevalent as product accumulates, these phenomena were also taken into account in all rate equations (Equations S1, equations 2 & 5?). For inhibited reaction progress curves, the same approach was applied on the rate laws for the three common types of inhibition (Equations S1, equations 5?). Additional equations used in the simulation tool are shown in Equations S1 (equations 8?3). The underlying assumption is that the rapid equilibrium approximations holds true throughout the complete reaction and that the reactions are pseudo-first-order in [S].
Figure 5. Screen dump of a subsection of the simulation tool. The tool contains three blocks (one for competitive, one for uncompetitive, and one for mixed inhibition; non-competitive inhibition is achieved by setting the two Ki values for mixed inhibition to equal values) in which the reaction parameters and variables can be set. The block for mixed inhibition is shown. Adjustable values are shown in red and are found in the second row of each block. The identities of the adjustable values are shown in the top row. The resulting IC50 value and overall equilibrium constant of the reversible reaction are also shown in the top row. A table presents the time point of Dmax[P] and the associated key data that result from adjustment of reaction conditions. To deduce the corresponding data at other time points, the desired values can be entered into cells of the top row of the table. In the simulation tool, changes of reaction conditions are also visualized in various graphs.
violated as [S] approaches [E], the associated error is normally small (Fig. S5). Moreover, HTS assays are typically performed with [E] at orders of magnitude lower than [S]. A level with [S] approaching [E] is thus equivalent with almost complete substrate depletion and will therefore contribute very weakly to the overall reaction rate. For the reversed reaction, the equivalent condition is most pronounced at the very beginning of the reaction. However, since the product level at this stage is very low the overall contribution to the reaction rate will be small. Another reason to work with very low [E] is imposed by the fact that IC50 values lower than one-half of the [E] cannot be measured. Since compounds are typically screened at 1?00 mM it is common to work with [E] around 100 nM, which in most cases is much lower than [S]. Furthermore, since many enzyme systems deviate from MM kinetics (e.g. due to substrate inhibition or activation, random pathways, or allosteric effects) [17] and because accuracy is limited by experimental noise, the limitations of underlying models can be neglected. More sophisticated methods for numerical integration are also not justified for the same reasons. For instance, integration using the Runge-Kutta method only generated extremely small differences, compared to the method applied, that definitely are within the limits of experimental noise.inhibitor of presequence peptidase. Progress curves with and without bestatin (500 mM) was therefore collected. Other reaction constituents were: 50 mM HEPES pH 8.2, 20 mM substrate, 25 nM enzyme and 10 mM MgCl2. Substrate was added last to start the reactions. For presequence peptidase, a set of initial velocity reaction experiments with substrate concentration ranging from 1?0 mM was also performed. Other conditions were as for the progress curve experiment.Fitting of models to experimental dataTo perform data fitting, the sum of the squared errors between model and data were minimized using the Solver add-in bundled with excel [20]. This allows automatic minimization of a target cell containing the sum of the squared errors by changing the values of cells containing model parameters.
Abstract
In the previous study, we unraveled the unique “erasure strategy” during the mouse spermiogenesis. Chromatin associated proteins sequentially disassociated from the spermatid chromosome, which led to the termination of transcription in ?elongating spermatids. By this process, a relatively naive paternal chromatin was generated, which might be essential for the zygotic development. We supposed the regulation of histone acetylation played an important role throughout this “erasure” process. In order to verify this hypothesis, we treated mouse spermatids in vitro by histone acetylase (HAT) inhibitor Curcumin. Our results showed an inhibiting effect of Curcumin on the growth of germ cell line in a dosedependent manner. Accordingly, the apoptosis of primary haploid spermtids was increased by Curcumin treatment. As expected, the acetylated histone level was downregulated. Furthermore, we found the transcription in spermatids ceased in advance, the dynamics of chromatin associated factors was disturbed by Curcumin treatment. The regulation of histone acetylation should be one of the core reprogramming mechanisms during the spermiogenesis. The reproductive toxicity of Curcumin needs to be thoroughly investigated, which is crucial for its further clinical application.
Citation: Xia X, Cai H, Qin S, Xu C (2012) Histone Acetylase Inhibitor Curcumin Impairs Mouse Spermiogenesisn In Vitro Study. PLoS ONE 7(11): e48673. doi:10.1371/journal.pone.0048673 Editor: Qing-Yuan Sun, Institute of Zoology, Chinese Academy of Sciences, China Received June 14, 2012; Accepted September 28, 2012; Published November 7, 2012 Copyright: ?2012 Xia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
