Adenylate cyclase activity of the corpus luteum during the oestrous cycle of the pig

Basal adenylate cyclase values for corpora lutea (CL) removed from cyclic gilts on Days 3, 8, 13 and 18 were 178 +/- 61, 450 +/- 46, 220 +/- 25 and 208 +/- 18 pmol cAMP formed/min/mg protein, respectively. Basal activity was significantly elevated on Day 8 (P less than 0.001). LH-stimulatable adenylate cyclase values for CL from Days 3, 8, 13 and 18 were 242 +/- 83, 598 +/- 84, 261 +/- 27 and 205 +/- 17 pmol cAMP formed/min/mg protein respectively. Serum progesterone concentrations of 12 gilts bled every 2 days through one complete oestrous cycle ranged from 1.1 to 26.9 ng/ml with highest values between Days 8 and 12. The decline in serum progesterone concentrations was coincident with the decrease in basal adenylate cyclase activity. There was no LH-stimulatable adenylate cyclase activity present in the CL at the specific times of the oestrous cycle examined. We conclude that progesterone secretion by the pig CL is apparently dependent on basal activity of adenylate cyclase.     

Conditions leading to precipitation of ribulose bisphosphate carboxylase-oxygenase differ from those leading to enzyme activation

The relation between conditions leading to precipitation and/or activation of Ru-P₂ carboxylase have been explored in order to test the hypothesis that conformational changes leading to precipitation might be identical to those which are presumed to lead to enzyme activation. From the results of kinetic and solubility studies, we conclude that this hypothesis is not valid, since changes in solubility of Ru-P₂ carboxylase occur ten times as fast as changes in enzyme kinetics.Abbreviations Ru-P₂ ribulose 1,5-bisphosphate - PVP polyvinylpyrrolidone - DTE dithioerythritol - Bicine N,N-bishydroxy-2-ethylglycine     

Defining window-boundaries for genomic analyses using smoothing spline techniques

Background

High-density genomic data is often analyzed by combining information over windows of adjacent markers. Interpretation of data grouped in windows versus at individual locations may increase statistical power, simplify computation, reduce sampling noise, and reduce the total number of tests performed. However, use of adjacent marker information can result in over- or under-smoothing, undesirable window boundary specifications, or highly correlated test statistics. We introduce a method for defining windows based on statistically guided breakpoints in the data, as a foundation for the analysis of multiple adjacent data points. This method involves first fitting a cubic smoothing spline to the data and then identifying the inflection points of the fitted spline, which serve as the boundaries of adjacent windows. This technique does not require prior knowledge of linkage disequilibrium, and therefore can be applied to data collected from individual or pooled sequencing experiments. Moreover, in contrast to existing methods, an arbitrary choice of window size is not necessary, since these are determined empirically and allowed to vary along the genome.

Results

Simulations applying this method were performed to identify selection signatures from pooled sequencing F_ST data, for which allele frequencies were estimated from a pool of individuals. The relative ratio of true to false positives was twice that generated by existing techniques. A comparison of the approach to a previous study that involved pooled sequencing F_ST data from maize suggested that outlying windows were more clearly separated from their neighbors than when using a standard sliding window approach.

Conclusions

We have developed a novel technique to identify window boundaries for subsequent analysis protocols. When applied to selection studies based on F_ST data, this method provides a high discovery rate and minimizes false positives. The method is implemented in the R package GenWin, which is publicly available from CRAN.     

Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.     

Assessing the influence of the carbon oxidation-reduction state on organic pollutant biodegradation in algal-bacterial photobioreactors

The influence of the carbon oxidation–reduction state (CORS) of organic pollutants on their biodegradation in enclosed algal–bacterial photobioreactors was evaluated using a consortium of enriched wild-type methanotrophic bacteria and microalgae. Methane, methanol and glucose (with CORS −4, −2 and 0, respectively) were chosen as model organic pollutants. In the absence of external oxygen supply, microalgal photosynthesis was not capable of supporting a significant methane and methanol biodegradation due to their high oxygen demands per carbon unit, while glucose was fully oxidized by photosynthetic oxygenation. When bicarbonate was added, removal efficiencies of 37 ± 4% (20 days), 65 ± 4% (11 days) and 100% (2 days) were recorded for CH_4, CH₃OH and C₆H₁₂O₆, respectively due to the additional oxygen generated from photosynthetic bicarbonate assimilation. The use of NO₃⁻ instead of NH₄⁺ as nitrogen source (N oxidation–reduction state of +5 vs. −3) resulted in an increase in CH₄ degradation from 0 to 33 ± 3% in the absence of bicarbonate and from 37 ± 4% to 100% in the presence of bicarbonate, likely due to a decrease in the stoichiometric oxygen requirements and the higher photosynthetic oxygen production. Hypothetically, the CORS of the substrates might affect the CORS of the microalgal biomass composition (higher lipid content). However, the total lipid content of the algal–bacterial biomass was 19 ± 7% in the absence and 16 ± 2% in the presence of bicarbonate.     

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.