Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences

How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.     

Sacculatane diterpenoids from axenic cultures of the liverwort Fossombronia wondraczekii

Five new sacculatane diterpenoids, 17,18-epoxy-7-sacculaten-12,11-olide, 7,17-sacculatadien-11,12-olide, 11beta,12-epoxy-7,17-sacculatadien-11alpha-ol, 1beta-acetoxy-11beta,12-epoxy-7,17-sacculatadien-11alpha-ol and 1beta,15xi-diacetoxy-11,12-epoxy-8(12),9(11),17-sacculatatriene along with sacculatal and sacculatanolide have been isolated from axenic cultures of the liverwort Fossombronia wondraczekii and their structures assigned on the basis of their spectroscopical properties.     

Gemfibrozil and its oxidative metabolites: quantification of aglycones, acyl glucuronides, and covalent adducts in samples from preclinical and clinical kinetic studies

A gradient reversed-phase HPLC analysis for the direct measurement of gemfibrozil (GEM) and four oxidative metabolites in plasma and urine of humans and in tissue homogenates of rats was developed. The corresponding acyl glucuronides and the covalently bound protein adducts (in protein precipitates) were determined after liberation from the respective conjugates via alkaline hydrolysis. The limits of detection for the covalent adducts in human plasma are: 10 ng ml⁻¹ (GEM), 20 ng ml⁻¹ (M1), 0.5 ng ml⁻¹ (M2, M4), and 5 ng ml⁻¹ (M3). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy. It has been applied to the analysis of preclinical and clinical studies. Pharmacokinetic profiles of gemfibrozil, its metabolites, and covalent adducts in human plasma and rat tissue homogenates are given.     

Elimination Or Reduction of Wrinkles in Semithin Epoxy Sections by Vacuum Drying

Vacuum drying, under appropriate conditions, diminishes the warping and buckling of epoxy semithin sections and enhances visualization with light microscopy. Treatment of sections with chloroform or variations in the drying times or temperatures did not reduce wrinkling.     

Insights into sucrose isomerization from crystal structures of the Pseudomonas mesoacidophila MX-45 sucrose isomerase,MutB

Three-dimensional structures of a sucrose isomerase from Pseudomonas mesoacidophila MX-45, forming mainly trehalulose have been solved to resolutions in the range 1.8–2.2 Å. Native and mutant complexes give, for the first time, a thorough insight into substrate binding and recognition, and product specificities of these enzymes. This study has pinpointed essential residues for binding the substrate sucrose, and hereby given detailed information on the interactions between the enzyme active site and glucosyl- and fructosyl moieties. Moreover, the 3-D structures revealed an aromatic clamp formed by two phenylalanines, which plays an essential role in recognition of the substrate and in controlling the reaction specificity.     

A substrate-independent TR-FRET histone deacetylase inhibitor assay

Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor? 647-labeled HDAC inhibitor or "tracer," which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.     

Ulk1-mediated phosphorylation of AMPK constitutes a negative regulatory feedback loop

Unc-51-like kinase 1 (Ulk1) plays a central role in autophagy induction. It forms a stable complex with Atg13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200). This complex is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) in a nutrient-dependent way. AMP-activated protein kinase (AMPK), which is activated by LKB1/Strad/Mo25 upon high AMP levels, stimulates autophagy by inhibiting mTORC1. Recently, it has been described that AMPK and Ulk1 interact and that the latter is phosphorylated by AMPK. This phosphorylation leads to the direct activation of Ulk1 by AMPK bypassing mTOR-inhibition. Here we report that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity. Thus, we propose that Ulk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagy. In our model, phosphorylation of AMPK by Ulk1 represents a negative feedback circuit.