Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp

The gene encoding a peptidase that belongs to the proteinase K family of serine peptidases has been identified from a psychrotrophic Serratia sp., and cloned and expressed in Escherichia coli. The gene has 1890 base pairs and encodes a precursor protein of 629 amino acids with a theoretical molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains. The enzyme is recombinantly expressed as an active approximately 56 kDa peptidase and includes both C-terminal domains. Purified enzyme is converted to the approximately 34 kDa form by autolytic cleavage when incubated at 50 degrees C for 30 min, but retains full activity. In the present work, the Serratia peptidase (SPRK) is compared with the family representative proteinase K (PRK) from Tritirachium album Limber. Both enzymes show a relatively high thermal stability and a broad pH stability profile. SPRK possess superior stability towards SDS at 50 degrees C compared to PRK. On the other hand, SPRK is considerably more labile to removal of calcium ions. The activity profiles against temperature and pH differ for the two enzymes. SPRK shows both a broader pH optimum as well as a higher temperature optimum than PRK. Analysis of the catalytic properties of SPRK and PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as substrate showed that SPRK possesses a 3.5-4.5-fold higher kcat at the temperature range 12-37 degrees C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat/Km) of SPRK compared to PRK.     

Mathematical modeling identifies inhibitors of apoptosis as mediators of positive feedback and bistability

The intrinsic, or mitochondrial, pathway of caspase activation is essential for apoptosis induction by various stimuli including cytotoxic stress. It depends on the cellular context, whether cytochrome c released from mitochondria induces caspase activation gradually or in an all-or-none fashion, and whether caspase activation irreversibly commits cells to apoptosis. By analyzing a quantitative kinetic model, we show that inhibition of caspase-3 (Casp3) and Casp9 by inhibitors of apoptosis (IAPs) results in an implicit positive feedback, since cleaved Casp3 augments its own activation by sequestering IAPs away from Casp9. We demonstrate that this positive feedback brings about bistability (i.e., all-or-none behaviour), and that it cooperates with Casp3-mediated feedback cleavage of Casp9 to generate irreversibility in caspase activation. Our calculations also unravel how cell-specific protein expression brings about the observed qualitative differences in caspase activation (gradual versus all-or-none and reversible versus irreversible). Finally, known regulators of the pathway are shown to efficiently shift the apoptotic threshold stimulus, suggesting that the bistable caspase cascade computes multiple inputs into an all-or-none caspase output. As cellular inhibitory proteins (e.g., IAPs) frequently inhibit consecutive intermediates in cellular signaling cascades (e.g., Casp3 and Casp9), the feedback mechanism described in this paper is likely to be a widespread principle on how cells achieve ultrasensitivity, bistability, and irreversibility.     

Seasonal plankton dynamics along a cross-shelf gradient

Much interest has recently been devoted to reconstructing the dynamic structure of ecological systems on the basis of time-series data. Using 10 years of monthly data on phyto- and zooplankton abundance from the Bay of Biscay (coastal to shelf-break sites), we demonstrate that the interaction between these two plankton components is approximately linear, whereas the effects of environmental factors (nutrients, temperature, upwelling and photoperiod) on these two plankton population growth rates are nonlinear. With the inclusion of the environmental factors, the main observed seasonal and inter-annual dynamic patterns within the studied plankton assemblage also indicate the prevalence of bottom-up regulatory control.     

Good reindeer mothers live longer and become better in raising offspring

Longevity is the main factor influencing individual fitness of long-lived, iteroparous species. Theories of life history evolution suggest this is because increased longevity allows individuals to (i) have more breeding attempts (time component), (ii) accumulate experience so as to become better able to rear offspring (experience component) or (iii) because individuals reaching old age have above-average quality (quality component). We assess empirically the relative influences of time, experience and quality on the relationship between longevity and individual fitness among female reindeer. Fitness increased with longevity due to all three processes. All females increased in success with age up to their penultimate year of life (experience component), the success of the terminal-breeding occasion was strongly dependent on longevity. Long-lived females had more successful breeding attempts during their life (time component), and had higher reproductive success at all ages, especially during the last year of life (individual quality component) than short-lived females. Our study reveals a more complex relationship between longevity and fitness in large mammals than the simple increase of the number of reproductive attempts when living longer.     

Propensity of a circadian clock to adjust to the 24h day-night light cycle and its sensitivity to molecular noise

The circadian clock of Drosophila melanogaster and its tendency to adjust to the day-night light cycle is simulated by deterministic and stochastic methods. The robustness of the locking to the light-cycle with respect to molecular noise is studied. It is found that within the model studied, the molecular noise in the stochastic simulation erases the finer injection-locking structures, stronger injection signals are needed and the locking has the character of prolonged locked time intervals with cycle slips in between. The simulations are compared to a simple injection-locking model with noise that seems to describe the overall behavior well.     

Environmental fluctuations facilitate species co-existence and increase decomposition in communities of wood decay fungi

Correlations between mast fruiting of bilberry Vaccinium myrtillus and peak levels of Clethrionomys-voles have been reported from both Norway and Finland, but there has been a discussion whether this is a bottom-up or a top-down relationship. In a multiple regression model, 65% of the variation in a bilberry production index calculated from game reports from southern Norway 1932–1977 could be explained by the berry index of the two preceding years and climate factors acting during key stages of the flowering cycle. High vole populations in previous years did not contribute to explain the fluctuation in berry production. I used the selected model and climate data to predict bilberry production for the period 1978–2004. Predicted berry indices of the current and previous year explained 38% and the total amount of precipitation in May–June explained 16% of the variation in a log-transformed snap-trapping index of bank vole Clethrionomys glareolus 1980–2004. The vole index was not related to any of the climate variables used to predict berry production. This pattern supports the hypothesis that vole cycles are generated by changes in plant chemistry due to climate-synchronized mast fruiting.     

A systematic evaluation of different hydrogen bonding patterns in unsymmetrical 1,n′-disubstituted ferrocenoyl peptides

The synthesis and spectroscopic characterization of 21 l,l′-disubstituted ferrocenoyl peptides of the general formula [Fe(C₅H₄-CO-Aal-OR) (C₅H₄-CO-Aa2-OR′)] is reported, with Aal and Aa2 being different amino acids. The one-pot synthesis from activated ferrocene-l,l′-dicarboxylic acid and two different amino acid esters gives the unsymmetrical ferrocenoyl peptides in yields between 27% and 42%, which can be easily separated from their symmetrical byproducts by column chromatography. All new compounds are comprehensively characterized by mass spectrometry (El and FAB, including high-resolution EI-MS), ¹H and ¹³C NMR, and UV/Vis spectroscopy. CD spectroscopy in conjunction with ¹H NMR is used to elucidate the solution structures. Using the achiral glycine (Gly) as Aal permits to determine qualitatively the structure-determining influence of the different amino acids Aa2. Helically chiral structures in ferrocene amino acids in this study are stabilized by hydrogen bonds. If one hydrogen bond partner is systematically moved away by the introduction of methylene groups, then indeed the strength of the hydrogen bond decreases as indicated by ¹H NMR chemical shifts of the amide protons and the strength of characteristic CD bands. As proline (Pro) is the only naturally accuring secondary amino acid it cannot contribute any amide proton to intra-strand hydrogen bonding. DFT calculations on the compound [Fe(C₅H₄-CO-Gly-OMe)(C₅H₄-CO-Pro-OMe)] with one achiral and one secondary amino acid were therefore performed to quantify the more subtle influence of the relative orientations of the ferrocene carbonyl groups and the cis-/trans-conformation of both amide bonds. Not unexpectedly, the conformations with both amide bonds in cis orientation are highest in energy. Surprisingly, the calculations suggest the presence of a low-energy conformation with a non-classical hydrogen bond between the proline ester carbonyl oxygen and a glycine H_α atom. However, a second conformation with no apparent intra-strand contacts but optimal positioning of all relevant groups is similar in energy. Although two conformations were observed in solution for this compound, the experimental data did not permit to assign those two conformations.     

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75?, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90?) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55?) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1?) and Mn,Fe(III)Fe(II) species (~3.3-3.4?) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.