A novel phosphatidylinositol manno-oligosaccharide (dPIM-8) from Gordonia sputi

The deacylated phosphatidylinositol manno-oligosaccharides (dPIMs) from the glycosyl phosphatidylinositol (GPI) carbohydrate antigen anchor of Gordonia sputi were the known 2,6-di-O-alpha-mannopyranosyl-myo-inositol glycerophosphate (dPIM-2) and the illustrated novel compound (dPIM-8), which could not be separated from dPIM-7 and dPIM-6, these three compounds being present in the mixture in the molar ratios 1.0:0.65:0.4. dPIM-8 is an analogue of dPIM-2 (and also of dPIM-7 and dPIM-6) in having alpha-mannopyranose and an alpha-mannopyranosyl linked heptasaccharide bonded to O-2 and O-6, respectively, of the inositol. The dPIM-8 species has not been found previously. [structure: see text]     

The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation

UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48- and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.     

Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase

Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5'-thymine of a thymine-thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60 degrees in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5'-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.     

Estimating the influence of population density and dispersal behavior on the ability to detect and monitor Agrilus planipennis (Coleoptera: Buprestidae) populations

Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), a phloem-feeding pest of ash (Fraxinus spp.) trees native to Asia, was first discovered in North America in 2002. Since then, A. planipennis has been found in 15 states and two Canadian provinces and has killed tens of millions of ash trees. Understanding the probability of detecting and accurately delineating low density populations of A. planipennis is a key component of effective management strategies. Here we approach this issue by 1) quantifying the efficiency of sampling nongirdled ash trees to detect new infestations of A. planipennis under varying population densities and 2) evaluating the likelihood of accurately determining the localized spread of discrete A. planipennis infestations. To estimate the probability a sampled tree would be detected as infested across a gradient of A. planipennis densities, we used A. planipennis larval density estimates collected during intensive surveys conducted in three recently infested sites with known origins. Results indicated the probability of detecting low density populations by sampling nongirdled trees was very low, even when detection tools were assumed to have three-fold higher detection probabilities than nongirdled trees. Using these results and an A. planipennis spread model, we explored the expected accuracy with which the spatial extent of an A. planipennis population could be determined. Model simulations indicated a poor ability to delineate the extent of the distribution of localized A. planipennis populations, particularly when a small proportion of the population was assumed to have a higher propensity for dispersal.     

Plume characteristics and dynamics of UV and IR laser-desorbed oligonucleotides

Laser desorption of dye-tagged oligonucleotides was studied using laser-induced fluorescence imaging. Desorption with ultra violet (UV) and infra-red (IR) lasers resulted in forward directed plumes of molecules. In the case of UV desorption, the initial shot desorbed approximately seven-fold more material than subsequent shots. In contrast, the initial shot in IR desorption resulted in the ejection of less material compared to subsequent shots and these plumes had a component directed along the path of the laser. Thermal equilibrium of the molecules in the plume was achieved after approximately 25 μs with a spread in molecular temperature which was described by a modified Maxwell-Boltzmann equation.     

Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.     

Regulation of DNA glycosylases and their role in limiting disease

This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations.