Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase/oxidase

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline, L-pipecolic acid and sarcosine, with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD, FMN and ATP per alphabeta complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline, for which a bell-shaped dependence (pK(a) values 7.0 +/- 0.2 and 7.6 +/- 0.2) for k(cat)/K(m) as a function of pH was observed. The pH dependence of k(cat) is sigmoidal, described by a single macroscopic pK(a) of 7.7 +/- 0.1, tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 +/- 0.05 mV x C degrees (-1)), and extrapolation to physiologic growth temperature for P. furiosus (100 degrees C) yields a value of - 407 +/- 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic L-proline dehydrogenase. Implications for its mechanism of action are discussed.     

Correction to: Managing biological invasions: the cost of inaction

Biological Invasions -     

Phenological mismatch drives selection on elevation,but not on slope,of breeding time plasticity in a wild songbird

Phenotypic plasticity is an important mechanism for populations to respond to fluctuating environments, yet may be insufficient to adapt to a directionally changing environment. To study whether plasticity can evolve under current climate change, we quantified selection and genetic variation in both the elevation (RN_E) and slope (RN_S) of the breeding time reaction norm in a long‐term (1973–2016) study population of great tits (Parus major). The optimal RN_E (the caterpillar biomass peak date regressed against the temperature used as cue by great tits) changed over time, whereas the optimal RN_S did not. Concordantly, we found strong directional selection on RN_E, but not RN_S, of egg‐laying date in the second third of the study period; this selection subsequently waned, potentially due to increased between‐year variability in optimal laying dates. We found individual and additive genetic variation in RN_E but, contrary to previous studies on our population, not in RN_S. The predicted and observed evolutionary change in RN_E was, however, marginal, due to low heritability and the sex limitation of laying date. We conclude that adaptation to climate change can only occur via micro‐evolution of RN_E, but this will necessarily be slow and potentially hampered by increased variability in phenotypic optima.     

Evaluating the genetic structure of wild and commercial red cusk-eel (Genypterus chilensis) populations through the development of novel microsatellite markers from a reference transcriptome

Molecular Biology Reports - The red cusk-eel (Genypterus chilensis) is a native Chilean species with a high-value market, with the potential to diversify Chilean aquaculture. The objective of this...     

Resource pulses affect prey selection and reduce dietary diversity of dingoes in arid Australia

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Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor γ chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-γ clones, and lysis of erbB-2⁺ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-γ, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL. Received: 23 July 1998 / Accepted: 5 October 1998     

Contribution to the Biology of the Vermilion Snapper, Rhomboplites aurorubens, in Trinidad and Tobago, West Indies

Reproduction and growth of the vermilion snapper, Rhomboplites aurorubens, were studied in Trinidad and Tobago. The smallest individual caught measured 145mm total length (TL) and all fish appeared to be mature. It was not possible to precisely determine size at first maturity due to the use of macroscopic techniques. The smallest spent male and female measured 181 and 211mm TL respectively, suggesting a size at first maturity below these sizes. Spawning occurred throughout the year, with a period of peak spawning from about June to November in the rainy season when river runoff increased. Sagittal otolith sections were used for age determination and the opaque ring, which was counted as the annulus, was deposited from January to May in the dry season. A total of 11 age groups between the ages of 2–12 years (155–505mm total length) were found. The von Bertalanffy growth parameters were: L=532mm, K=0.13y^–1, and t₀=–0.17, where L is the asymptotic length, K is the growth coefficient and t₀ is the theoretical age at zero length. The relationship between weight (WT) and length (TL) was WT=3.43×10^–5 TL^2.82. Vermilion snapper in this study area appears to grow slower and attain a smaller asymptotic length, but has a longer lifespan than found in populations in higher latitudes. This may be attributed to different levels of exploitation, which may be higher in the latter areas.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust