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Size Fractionation of Titania Nanoparticles in Wild Dittrichia viscosa Grown in a Native Environment

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-06-15
Abstract: 

We report a size fractionation of titania (TiO2) nanoparticles absorbed from the environment and found within wild Dittrichia viscosa plants. The nanoparticles were isolated by extraction and isolation from distinct plant organs, as well as from the corresponding rhizosphere of wild, adult plants. The collected nanoparticles were characterized by scanning transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (STEM-EDS). More than 1,200 TiO2 nanoparticles were analyzed by these techniques. The results indicated the presence of TiO2 nanoparticles with a wide range of sizes within the inspected plant organs and rhizospheres. Interestingly, a size selective process occurs during the internalization and translocation of these nanoparticles (e.g., foliar and root uptake), which favors the accumulation of mainly TiO2 nanoparticles with diameters <50 nm in the leaves, stems, and roots. In fact, our findings indicate that among the total number of TiO2 nanoparticles analyzed, the fraction of the particles with dimensions <50 nm were 52 of those within the rhizospheres, 88.5% of those within the roots, 90% of those within the stems, and 53% of those within the leaves. This significant difference observed in the size distribution of the TiO2 nanoparticles among the rhizosphere and the plant organs could have impacts on the food chain, and further biologicals effects that are dependent on the size of the TiO2.

Document type: 
Article

Identification of Asp174 and Asp175 as the Key Catalytic Residues of Human O-GlcNAcase by Functional Analysis of Site-Directed Mutants

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2006-02-24
Abstract: 

O-GlcNAcase is a family 84 â-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of â-O-linked 2-acetamido-2-deoxy-D-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp174 as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp175 as the residue that functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.

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Analysis of PUGNAc and NAG-thiazoline as Transition State Analogues for Human O-GlcNAcase:  Mechanistic and Structural Insights into Inhibitor Selectivity and Transition State Poise

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2006-12-23
Abstract: 

O-GlcNAcase catalyzes the cleavage of β-O-linked 2-acetamido-2-deoxy-β-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2‘-methyl-α-d-glucopyranoso[2,1-d]-Δ2‘-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal β-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme−inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.

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Article
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A highly concise preparation of O-deacetylated arylthioglycosides of N-acetyl-D-glucosamine from 2-acetamido-3,4,6-tri-O-acetyl2-deoxy-a-D-glucopyranosyl chloride and aryl thiols or disulfides

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2006-07-01
Abstract: 

An expedient and mild route to a range of aryl 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranosides has been devised from 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosyl chloride and arylthiols or aryl disulfides using phase transfer catalysis conditions. This simple procedure compresses up to three synthetic steps into a one-pot reaction, obviating the need for tedious workups and chromatography and directly furnishes crystalline materials in good yields. The procedure is compatible with a range of thiols and disulfides and may be amenable for preparing a wide range of thioglycosides with various glycons and aglycons.

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Article
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O-GlcNAcase Catalyzes Cleavage of Thioglycosides without General Acid Catalysis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005-11-16
Abstract: 

O-GlcNAcase catalyzes the removal of N-acetylglucosamine residues from serine and threonine residues of post-translationally modified proteins using a catalytic mechanism involving substrate-assisted catalysis and general acid/base catalysis. Since thioglycosides are widely perceived as resistant to hydrolysis by glycosidases, it was surprising to find that O-GlcNAcase also catalyzes the efficient hydrolysis of S-glycosides. Brønsted analyses and pH-activity studies of the O-GlcNAcase-catalyzed hydrolysis of a series of aryl S- and O-glycosides reveal that O-GlcNAcase effects hydrolysis of thioglycosides without the assistance of general acid catalysis. α-Deuterium kinetic isotope effects for O- and S-glycosides, as well as Taft-like analyses using N-fluoroacetyl-β-glycosides, suggest that O-GlcNAcase accomplishes hydrolysis of thioglycosides by stabilizing late transition states. For S-glycosides this transition state shows greater nucleophilic participation from the 2-acetamido group than for O-glycosides. The rate constants governing the O-GlcNAcase-catalyzed hydrolysis of O- and S-glycosides as compared to those previously determined for the spontaneous hydrolysis of structurally similar O,O- and O,S-acetals show a similar ratio. O-GlcNAcase therefore demonstrates similar catalytic proficiency toward both O- and S-glycosides. We conclude that O-GlcNAcase is a bifunctional catalyst capable of efficiently cleaving thioglycosides without general acid catalysis, an observation that may have biological implications.

Document type: 
Article

Free Radical Chemistry of Phosphasilenes

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-06-02
Abstract: 

Understanding the characteristics of radicals formed from silicon-containing heavy analogues of alkenes is of great importance for their application in radical polymerization. Bulky and electronic substituent effects in such compounds as phosphasilenes not only stabilize the Si=P double bond, but also influence the structure and species of the formed radicals. Herein we report our first investigations of radicals derived from phosphasilenes with Mes (2,4,6-trimethylphenyl), Tip (2,4,6-triisopropylphenyl), Dur (2,3,5,6-tetramethylphenyl) and NMe2 (dimethylamino) substituents on the P atom, using muon spin spectroscopy and DFT calculations. Adding muonium (a light isotope of hydrogen) to phosphasilenes reveals that: a) the electron-donor NMe2 and the bulkiest Tip-substituted phosphasilenes form several muoniated radicals with different rotamer conformations; b) bulky Dur-substituted phosphasilene forms two radicals (Si- and P-centred); and c) Mes-substituted phosphasilene mainly forms one species of radical, at the P centre. These significant differences result from intramolecular substituent effects.

Document type: 
Article

Multifunctional Compounds for Activation of the p53‐Y220C Mutant in Cancer

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-09-19
Abstract: 

The p53 protein plays a major role in cancer prevention, and over 50 % of cancer diagnoses can be attributed to p53 malfunction. The common p53 mutation Y220C causes local protein unfolding, aggregation, and can result in a loss of Zn in the DNA‐binding domain. Structural analysis has shown that this mutant creates a surface site that can be stabilized using small molecules, and herein a multifunctional approach to restore function to p53‐Y220C is reported. A series of compounds has been designed that contain iodinated phenols aimed for interaction and stabilization of the p53‐Y220C surface cavity, and Zn‐binding fragments for metallochaperone activity. Their Zn‐binding affinity was characterized using spectroscopic methods and demonstrate the ability of compounds L4 and L5 to increase intracellular levels of Zn2+ in a p53‐Y220C‐mutant cell line. The in vitro cytotoxicity of our compounds was initially screened by the National Cancer Institute (NCI‐60), followed by testing in three stomach cancer cell lines with varying p53 status’, including AGS (WTp53), MKN1 (V143A), and NUGC3 (Y220C). Our most promising ligand, L5, is nearly 3‐fold more cytotoxic than cisplatin in a large number of cell lines. The impressive cytotoxicity of L5 is further maintained in a NUGC3 3D spheroid model. L5 also induces Y220C‐specific apoptosis in a cleaved caspase‐3 assay, reduces levels of unfolded mutant p53, and recovers p53 transcriptional function in the NUGC3 cell line. These results show that these multifunctional scaffolds have the potential to restore wild‐type function in mutant p53‐Y220C.

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Metal Complexes that Bind to the Amyloid-β Peptide of Relevance to Alzheimer's Disease

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-03-10
Abstract: 

Alzheimer’s disease (AD) is the most common form of dementia, and is a multi-faceted disease that is characterized by oxidative stress, metal-ion dysregulation, and the formation of intracellular neurofibrillary tangles of tau protein and extracellular amyloid-β (Aβ) aggregates. This review will focus on the interaction of metal complexes with the Aβ peptide, and how these interactions can modify the peptide aggregation pathway, oxidative stress, and overall toxicity of the Aβ peptide. While certain endogenous metal complexes such as heme can enhance toxicity, a large number of reports detail the potentially protective effect of discrete metal complexes in AD. These results will be discussed in the context of ligand design to target specific peptide residues for covalent binding, modulate peptide aggregation towards non-toxic species, and enhance blood brain barrier access. Additional features of metal complexes such as light-activated Aβ binding, catalytic antioxidant activity, and peptidase activity will be detailed.

Document type: 
Article

Differentiation of Nanoparticles Isolated from Distinct Plant Species Naturally Growing in a Heavy Metal Polluted Site

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2019-11-08
Abstract: 

Leaves harvested from the plants of two different species (Dittrichia viscosa and Cichorium intybus) grown in their autogenous environment near a steel manufacturing company were characterized for naturally accumulated nanoparticles. These plant species are known to accumulate heavy metals. It was, however, unknown if these species would also accumulate these heavy metals in the form of nanoparticles. The isolated solid fractions were analyzed using dynamic light scattering, X-ray fluorescence, and transmission electron microscopy. These analyses revealed the presence of nanoparticles within the plants. The composition of nanoparticles found in each plant species is distinct: (i) for Dittrichia viscosa, the nanoparticle composition matched the heavy metal pollution anticipated from the surrounding industries; (ii) for Cichorium intybus, the nanoparticle composition was similar to the most abundant elements in the soil. The different behavior is a reflection of the phytoaccumulator characteristics of both species. This study provides the first evidence of sequestration of heavy metals in the form of nanoparticles by plants grown autogenously in polluted areas and will have implications in waste management of phytoremediation systems and in understanding the heavy metal life-cycle in the environment.

Document type: 
Article

A New Quinoline-Based Chemical Probe Inhibits the Autophagy-Related Cysteine Protease ATG4B

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-08-03
Abstract: 

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4–28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

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