Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.     

Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase

The primary structure of a region on hormone-sensitive lipase was determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser- Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Lys. Ser-8 was the only residue in the intact protein phosphorylated by cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo.     

Regulation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase

Phosphorylation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase results in activation of both cholesterol ester and triacylglycerol hydrolase activities. Activation against both substrates correlates closely with phosphorylation in time course experiments. Proteolytic digestion of phosphorylated cholesterol ester hydrolase, followed by peptide mapping, indicates the presence of a single phosphorylation site on the enzyme. Phosphoserine is the only phosphoamino acid detected following partial acid hydrolysis of the phosphorylated enzyme.     

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na(+)-independent and inhibited almost completely by 2-amino-endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na(+)-independent leucine transport (provisionally designated system bo,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system bo,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na(+)-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na(+)-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na(+)-dependent system Bo,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system Bo,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system Bo,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system Bo,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Rapid and accurate structure-based therapeutic peptide design using GPU accelerated thermodynamic integration

Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity, and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability.     

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.     

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.