Strategies for modulating the pH-dependent activity of a family 11 glycoside hydrolase.

The pH-dependent activity of wild-type Bacillus circulans xylanase (BcX) is set by the pK(a) values of its nucleophile Glu78 and general acid/base Glu172. Herein, we examined several strategies to manipulate these pK(a) values and thereby shift the pH(opt) at which BcX is optimally active. Altering the global charge of BcX through random succinylation had no significant effect.

Paraneoplastic hypertrophic osteopathy in 30 dogs.

Paraneoplastic hypertrophic osteopathy (pHO) is known to occur in both canine and human cancer patients. While the pathology of pHO is well-described in the dog, very little information exists regarding the true clinical presentation of dogs affected with pHO. The primary objective of this study was to provide a more comprehensive clinical picture of pHO.

Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity.

Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro.

α-Galacturonidase(s): a new class of Family 4 glycoside hydrolases with strict specificity and a unique CHEV active site motif.

The catalytic activity of the Family 4 glycosidase LplD protein, whose active site motif is CHEV, is unknown despite its crystal structure having been determined in 2008. Here we identify that activity as being an α-galacturonidase whose natural substrate is probably α-1,4-di-galacturonate (GalUA2). Phylogenetic analysis shows that LplD belongs to a monophyletic clade of CHEV Family 4 enzymes, of which four other members are also shown to be galacturonidases.

Structure-based mutagenic analysis of mechanism and substrate specificity in mammalian glycosyltransferases: porcine ST3Gal-I.

Sialyltransferases (STs) play essential roles in signaling and in the cellular recognition processes of mammalian cells by selectively installing cell-surface sialic acids in an appropriate manner both temporally and organ-specifically. The availability of the first three-dimensional structure of a mammalian (GT29) sialyltransferase has, for the first time, allowed quantitative structure/function analyses to be performed, thereby providing reliable insights into the roles of key active site amino acids. Kinetic analyses of mutants of ST3Gal-I, in conjunction with structural studies, have confirmed the mechanistic roles of His302 and His319 as general acid and base catalysts, respectively, and have quantitated other interactions with the cytosine monophosphate-N-acetyl β-neuraminic acid donor substrate.

The 'mirror-image' postulate as a guide to the selection and evaluation of pyrrolidines as α-l-fucosidase inhibitors.

The ability of a series of pyrrolidines to inhibit several glycosidases was investigated. Using Fleet's 'mirror-image postulate', it was proposed that enantiomeric derivatives of 1,4-dideoxy-1,4-imino-d-lyxitol (a known α-d-galactosidase inhibitor) would show inhibitory activity against α-l-fucosidases. Some modest α-l-fucosidase inhibitory activity was observed for selected compounds (particularly an aminomethyl pyrrolidine) and it was proposed that better activity could be obtained by modifying the C-2 side chain of the pyrrolidine core.

Perivascular adipose tissue-derived adiponectin activates BK(Ca) channels to induce anticontractile responses.

This study aims to identify the potential mechanisms by which perivascular adipose tissue (PVAT) reduces tone in small arteries. Small mesenteric arteries from wild-type and large-conductance Ca(2+)-activated K(+) (BKCa) channel knockout mice were mounted on a wire myograph in the presence and absence of PVAT, and contractile responses to norepinephrine were assessed. Electrophysiology studies were performed in isolated vessels to measure changes in membrane potential produced by adiponectin.

Investigating the structural dynamics of α-1,4-galactosyltransferase C from Neisseria meningitidis by nuclear magnetic resonance spectroscopy.

Neisseria meningitidis α-1,4-galactosyltransferase C (LgtC) is responsible for the transfer of α-galactose from donor UDP-galactose to the lipooligosaccharide terminal acceptor lactose. Crystal structures of its substrate analogue complexes have provided key insights into the galactosyl transfer mechanism, including a hypothesized need for active site mobility. Accordingly, we have used nuclear magnetic resonance spectroscopy to probe the structural dynamics of LgtC in its apo form and with bound substrate analogues.

Isolated small uterine arteries from non-pregnant and term pregnant rats exhibit regulatory responses to elevations in extravascular pressure.

An adequate blood supply is essential for the maintenance of uterine function and fetal health during parturition. However, labouring uterine contractions will impart compressive forces on small uterine arteries (SUA). We demonstrate that isolated, pressurised rat SUA arteries, pre-constricted with arginine vasopressin or high potassium solution, exhibit regulatory responses to elevations in extravascular pressure (EVP) which maintain internal diameter constant at EVPs of 0-40 mm Hg.

The potential of bacteria isolated from ruminal contents of seaweed-eating North Ronaldsay sheep to hydrolyse seaweed components and produce methane by anaerobic digestion in vitro.

The production of methane biofuel from seaweeds is limited by the hydrolysis of polysaccharides. The rumen microbiota of seaweed-eating North Ronaldsay sheep was studied for polysaccharidic bacterial isolates degrading brown-seaweed polysaccharides. Only nine isolates out of 65 utilized >90% of the polysaccharide they were isolated on.

Detection of chromosome x;18 breakpoints and translocation of the xq22.3;18q23 regions resulting in variable fertility phenotypes.

We describe a familial pattern of gonosomal-autosomal translocation between the X and 18 chromosomes, balanced and unbalanced forms, in male and female siblings. The proposita was consulted for hypergonadotropic hypogonadism. Karyotype analysis revealed a balanced 46, X, t(X;18)(q22.

Order and disorder: differential structural impacts of myricetin and ethyl caffeate on human amylase, an antidiabetic target.

The increasing prevalence of diabetes has accelerated the search for new drugs derived from natural sources. To define the functional features of two such families of compounds, the flavonols and the ethyl caffeates, we have determined the high-resolution structures of representative inhibitors in complex with human pancreatic α-amylase. Myricetin binds at the active site and interacts directly with the catalytic residues despite its bulky planar nature.

Identification of Tyr241 as a key catalytic base in the family 4 glycoside hydrolase BglT from Thermotoga maritima.

While the vast majority of glycosidases catalyze glycoside hydrolysis via oxocarbenium ion-like transition states and typically employ carboxylic acid residues as acid/base or nucleophile catalysts, two subfamilies of these enzymes (GH4 and GH109 in the CAZY classification) conduct hydrolysis via a redox-assisted mechanism involving anionic transition states. While good evidence of this mechanism has been obtained, the identities of the catalytic residues involved have not yet been confirmed. Mechanistic analyses of mutants of the 6-phospho-β-glucosidase from Thermotoga maritima (BglT), in which the active site tyrosine residue (Tyr 241) has been replaced with Phe and Ala, provide support for its role as a catalytic base.

Nuclear magnetic resonance spectral assignments of α-1,4-galactosyltransferase LgtC from Neisseria meningitidis: substrate binding and multiple conformational states.

Lipopolysaccharide α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitidis is responsible for a key step in lipooligosaccharide biosynthesis involving the transfer of α-galactose from the sugar donor UDP-galactose to a terminal acceptor lactose. Crystal structures of the complexes of LgtC with Mn(2+) and the sugar donor analogue UDP-2-deoxy-2-fluorogalactose in the absence and presence of the sugar acceptor analogue 4'-deoxylactose provided key insights into the galactosyl-transfer mechanism. Combined with kinetic analyses, the enzymatic mechanism of LgtC appears to involve a "front-side attack" S(N)i-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate.

Evaluation of a single subcutaneous infusion of carboplatin as adjuvant chemotherapy for dogs with osteosarcoma: 17 cases (2006-2010).

Objective: To evaluate adverse effects and survival times in dogs with osteosarcoma that received a single SC infusion of carboplatin as adjunctive chemotherapeutic treatment following limb amputation or limb-sparing surgery.

Design: Retrospective case series.

Animals: 17 client-owned dogs with spontaneously occurring osteosarcoma.

Directed evolution of a β-glycosidase from Agrobacterium sp. to enhance its glycosynthase activity toward C3-modified donor sugars.

Glycans bearing modified hydroxyl groups are common in biology but because these modifications are added after assembly, enzymes are not available for the transfer and coupling of hydroxyl-modified monosaccharide units. Access to such enzymes could be valuable, particularly if they can also introduce 'bio-orthogonal tags'. Glycosynthases, mutant glycosidases that synthesize glycosides using glycosyl fluoride donors, are a promising starting point for creation of such enzymes through directed evolution.

A chemoenzymatic total synthesis of the neurogenic starfish ganglioside LLG-3 using an engineered and evolved synthase.

An LLG-3 oligosaccharide-fluoride can be assembled chemoenzymatically and readily coupled with various sphingosines by an engineered endoglycoceramidase glycosynthase. The lyso-ganglioside products are acylated to generate the individual isomers identified in the heterogeneous natural isolates, as well as modified glycosphingolipids.

Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR.

Separation of small molecules such as biotinylated baits from solutions of filamentous bacteriophage is achieved generally through polyethylene glycol precipitation of the phage and centrifugation prior to affinity selection or panning. This method is laborious and time-consuming and is accompanied frequently by significant loss of virions, especially when performed at low phage concentrations. Similarly, accurate quantitation of phage is performed typically by counting plaques, a method that is tedious, low-throughput, and not amenable easily to high titers.

Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981-2009).

Background: Congenital toxoplasmosis is a severe, life-altering disease in the United States. A recently developed enzyme-linked immunosorbent assay (ELISA) distinguishes Toxoplasma gondii parasite types (II and not exclusively II [NE-II]) by detecting antibodies in human sera that recognize allelic peptide motifs of distinct parasite types.

Methods: ELISA determined parasite serotype for 193 congenitally infected infants and their mothers in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS), 1981-2009.

Isolation of improved free fatty acid overproducing strains of via Nile red based high-throughput screening.

Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially-produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose.

Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition.

The rice BGlu1 β-D-glucosidase nucleophile mutant E386G is a glycosynthase that catalyzes the synthesis of cellooligosaccharides from α-d-glucopyranosyl fluoride (GlcF) donor and p-nitrophenyl (pNP) cellobioside (Glc2-pNP) or cello-oligosaccharide acceptors. When activity with other donors and acceptors was tested, the initial enzyme preparation cleaved pNP-β-D-glucopyranoside (Glc-pNP) and pNP-β-D-fucopyranoside (Fuc-pNP) to pNP and glucose and fucose, suggesting contamination with wild type BGlu1 β-glucosidase. The products from reaction of GlcF and Fuc-pNP included Fuc-β-(1→3)-Fuc-pNP, Glc-β-(1→3)-Fuc-pNP, and Fuc-β-(1→4)-Glc-β-(1→3)-Fuc-pNP, suggesting the presence of both wild type BGlu1 and its glycosynthase.

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum.

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities.