Glycosyltransferases (GTs) are ubiquitous enzymes that catalyze the assembly of glycoconjugates found throughout all kingdoms of nature. A longstanding problem is the rational design of probes that can be used to manipulate GT activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analogue that inhibits, with high potency both in vitro and in cells, the human GT responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analogue. Treatment of cells with the precursor decreases O-GlcNAc in a targeted manner with a single digit micromolar EC50. This approach to inhibition of GTs should be applicable to other members of this increasingly interesting superfamily of enzymes and enable their manipulation in a biological setting.

[1] Electrical resistivity measurements of polycrystalline iron have been performed at 5, 7, and 15 GPa and in the temperature range 293-2200 K by employing a four-wired method. The kinks in electrical resistivity associated with solid iron phase transitions and the solid to liquid transition were clearly observed upon increasing temperature. Geometry corrections due to volume variations with pressure and temperature were applied to the entire data set. High pressure and temperature thermal conductivity were calculated by fitting resistivity data through the Wiedemann-Franz law. The temperature dependences of electrical resistivity and thermal conductivity for a, g, and e solid iron have been determined at highpressure conditions. Our study provides the first experimental constraint on the heat flux conducted at Mercury's outmost core, estimated to be 0.29-0.36 TW, assuming an adiabatic core. Extrapolations of our data to Martian outer core conditions yield a series of heat transport parameters (e.g., electrical resistivity, thermal conductivity, and heat flux), which are in reasonable comparison with various geophysical estimates. Citation: Deng, L., C. Seagle, Y. Fei, and A. Shahar (2013), High pressure and temperature electrical resistivity of iron and implications for planetary cores, Geophys.

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The modification of N-glycans by ␣-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for ␣1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-␣1,6-mannosidase activity consistent with specificity for N-linked glycans having their ␣1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substratebound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (␣/␣) 6 -fold, recognize and hydrolyze the ␣1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-␣1,6-mannosidases.A feature of emerging importance to bacteria that colonize or infect humans is their capacity to process host glycans. Streptococcus pneumoniae is one notable human pathogen that relies on this ability for its full virulence (1). Among its known carbohydrate active virulence factors are NanA, StrH, BgaA, and EndoD. NanA Glycoside hydrolases, enzymes that break glycosidic bonds through a hydrolytic mechanism, are presently classified into 123-amino acid sequence based families (2). ␣-Mannosidases known to process N-glycans are found in families 38, 47, 76, 92, and 99. Very recent studies have shown the bacterial family 38 ␣-mannosidase from Streptococcus pyogenes (SpyGH38) to be a specific exo-␣1,3-mannosidase that is tolerant of the ␣1,6-branches in N-glycans (3). Analysis of family 92 glycoside hydrolases from the human gut symbiont Bacteroides thetaiotaomicron revealed an expanded repertoire of ␣-mannosidases (4). These enzymes displayed activity primarily toward ␣1,2-and ␣1,3-mannosidic linkages with some having low ␣1,6-mannosidase activity. In addition to the established ability of S. pneumoniae to exo-hydrolytically process the distal arms of complex glycans, which comprise sialic acid, galactose, and N-acetylglucosamine, consideration of additional putative carbohydrate-active enzymes found in this organism suggests it can partly degrade the mannose component of N-glycans using enzymes similar to those found in S. pyogenes and B. thetaiotaomicron. Through these observations it has become clear that some bacteria, possibly including S. pneumoniae, have the capacity to process the mannose component of N-glycans. A noteworthy gap, however, in the known bacterial N-glycan de...

AmpC hyperproduction is the most frequent mechanism of resistance to penicillins and cephalosporins in Pseudomonas aeruginosa and is driven by ampD mutations or the recently described inactivation of dacB, which encodes the nonessential penicillin-binding protein (PBP) PBP 4. Recent work showed that nagZ inactivation attenuates ␤-lactam resistance in ampD mutants. Here we explored whether the same could be true for the dacB mutants with dacB mutations alone or in combination with ampD mutations. The inactivation of nagZ restored the wild-type ␤-lactam MICs and ampC expression of PAO1 dacB and ampD mutants and dramatically reduced the MICs (for example, the MIC for ceftazidime dropped from 96 to 4 g/ml) and the level of ampC expression (from ca. 1,000-fold to ca. 50-fold higher than that for PAO1) in the dacB-ampD double mutant. On the other hand, nagZ inactivation had little effect on the inducibility of AmpC. The NagZ inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate attenuated the ␤-lactam resistance of the AmpChyperproducing strains, showing a greater effect on the dacB mutant (reducing the ceftazidime MICs from 24 to 6 g/ml) than the ampD mutant (reducing the MICs from 8 to 4 g/ml). Additionally, nagZ inactivation in the dacB mutant blocked the overexpression of creD (blrD), which is a marker of the activation of the CreBC (BlrAB) regulator involved in the resistance phenotype. Finally, through population analysis, we show that the inactivation of nagZ dramatically reduces the capacity of P. aeruginosa to develop ceftazidime resistance, since spontaneous mutants were not obtained at concentrations >8 g/ml (the susceptibility breakpoint) for the nagZ mutant but were obtained with wild-type PAO1. Therefore, NagZ is envisaged to be a candidate target for preventing and reverting ␤-lactam resistance in P. aeruginosa.

O‐Linked glycosylation of serine and threonine residues of nucleocytoplasmic proteins with N‐acetylglucosamine (O‐GlcNAc) residues is catalyzed by O‐GlcNAc transferase (OGT). O‐GlcNAc is conserved within mammals and is implicated in a wide range of physiological processes. Herein, we describe metabolic precursor inhibitors of OGT suitable for use both in cells and in vivo in mice. These 5‐thiosugar analogues of N‐acetylglucosamine are assimilated through a convergent metabolic pathway, most likely involving N‐acetylglucosamine‐6‐phosphate de‐N‐acetylase (NAGA), to generate a common OGT inhibitor within cells. We show that of these inhibitors, 2‐deoxy‐2‐N‐hexanamide‐5‐thio‐d‐glucopyranoside (5SGlcNHex) acts in vivo to induce dose‐ and time‐dependent decreases in O‐GlcNAc levels in various tissues. Decreased O‐GlcNAc correlates, both in vitro within adipocytes and in vivo within mice, with lower levels of the transcription factor Sp1 and the satiety‐inducing hormone leptin, thus revealing a link between decreased O‐GlcNAc levels and nutrient sensing in peripheral tissues of mammals.

The complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-β-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.

Apatite and silicate glasses share the same water content calibration curves in isotope modes where water was determined from the H− intensity regardless of multicollection or peak jumping. In contrast, the slope of apatite significantly differs from that of silicate glasses in element mode where OH− was counted for the water content.