[Show abstract][Hide abstract] ABSTRACT: Background
Aortic valve sclerosis (AVS) is a chronic, progressive disease involving lipid infiltration, inflammation, and tissue calcification. Despite its high prevalence, there are currently no clinically-approved pharmaceuticals for the management of AVS. The objective of the current study was to elucidate the effects of an angiotensin II type 1 receptor blocker, alone or in combination with statin therapy, on the progression of AVS.
Male New Zealand White rabbits were fed an atherogenic diet for a period of 12 months to induce AVS. Once disease was established, rabbits were block randomly assigned to receive either no treatment, olmesartan medoxomil, atorvastatin calcium, or a combination of both drugs for a period of 6 months. Disease progression was monitored in vivo using clinically-relevant magnetic resonance imaging and aortic valve cusps were examined ex vivo using histological and immunohistochemical methods.
Cusp thickness significantly increased (0.58 ± 0.03 versus 0.39 ± 0.03 mm for Cholesterol and Control, respectively; P <0.0001) and all classic hallmarks of disease progression — including lipid infiltration, inflammation, and tissue calcification — were observed after 12 months. Unfortunately, neither olmesartan medoxomil nor atorvastatin calcium were able to reverse or delay disease progression during the 6 month treatment period. However, several histological changes were observed in the valvular microenvironment.
The current study suggests that angiotensin receptor blockers, alone or in combination with statin therapy, may not be suitable for management of clinical AVS.
The Canadian journal of cardiology 09/2014; 30(9). DOI:10.1016/j.cjca.2013.12.027 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis of defined glycans enables us to further understand their roles in a biological context. While useful chemical methods have been developed for the synthesis of glycans, these typically require complex protection and deprotection steps along with challenging control of anomeric stereochemistry. Enzymatic methods offer an attractive alternative to chemical synthesis. In particular, the use of glycosynthases and thioglycoligases, classes of engineered glycoside hydrolases, offer an enticing approach to the stereo- and regioselective synthesis of glycans without the need for protecting groups. Herein we describe recent progress in the use of glycosynthases and thioglycoligases for the synthesis of glycans and glycopolymers.
[Show abstract][Hide abstract] ABSTRACT: Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6,144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyze 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy Database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially-derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.
Journal of Biotechnology 07/2013; 167(4). DOI:10.1016/j.jbiotec.2013.07.015 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DypB from Rhodococcus jostii RHA1 is a bacterial dye-decolorizing peroxidase (DyP) that oxidizes lignin and Mn(II). Three residues interact with the iron-bound solvent species in ferric DypB: Asn-246 and the conserved Asp-153 and Arg-244. Substitution of either Asp-153 or Asn-246 with alanine minimally affected the second order rate constant for Compound I formation (k(1) ∼ 10(5) M(-1)s(-1)) and the specificity constant (k(cat)/K(m)) for H(2)O(2). Even in the D153A/N246A double variant, these values were reduced less than 30-fold. However, these substitutions dramatically reduced the stability of Compound I (t(1/2) ∼ 0.13 s) as compared with the wild-type enzyme (540 s). By contrast, substitution of Arg-244 with leucine abolished the peroxidase activity, and heme iron of the variant showed a pH-dependent transition from high spin (pH 5) to low spin (pH 8.5). Two variants were designed to mimic the plant peroxidase active site: D153H, which was more than an order of magnitude less reactive with H(2)O(2), and N246H, which had no detectable peroxidase activity. X-ray crystallographic studies revealed that structural changes in the variants are confined to the distal heme environment. The data establish an essential role for Arg-244 in Compound I formation in DypB, possibly through charge stabilization and proton transfer. The principle roles of Asp-153 and Asn-246 appear to be in modulating the subsequent reactivity of Compound I. These results expand the range of residues known to catalyze Compound I formation in heme peroxidases.
[Show abstract][Hide abstract] ABSTRACT: Arterial calcification is a common complication of several disorders and is a strong predictor of mortality. The mechanism underlying arterial calcification is not fully understood and as such, no pharmaceutical therapies are currently available which impede its progression. The aim of this study was to investigate the effects of an angiotensin II (AngII) type 1 receptor blocker (ARB) on arterial calcification.
Male New Zealand White rabbits were fed an atherogenic diet to induce atherosclerosis and arterial calcification over a period of 12 weeks, with an ARB administered in the final 4 weeks. Using clinically relevant micro-computed tomography, we found that animals fed the atherogenic diet displayed extensive arterial calcification when compared with control. In contrast, administration of the ARB completely inhibited calcification (2.80 ± 1.17 vs. 0.01 ± 0.01% calcified tissue in cholesterol and ARB-treated, respectively; n = 6 and 5; P < 0.05). Calcified regions were characterized by up-regulation of bone morphogenetic protein 2, osteocalcin, and the AngII type 1 receptor and concomitant down-regulation of α-smooth muscle actin, consistent with a phenotypic switch from vascular to osteoblast-like cells.
These data provide the first evidence that angiotensin receptor blockade can inhibit arterial calcification by disrupting vascular osteogenesis and suggest that ARBs may be a novel treatment option for patients suffering from vascular calcification.
Cardiovascular Research 04/2011; 90(1):165-70. DOI:10.1093/cvr/cvq391 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site-saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.