Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

O‐GlcNAc (2‐acetamino‐2‐deoxy‐β‐D‐glucopyranose), an important modification for cellular processes, is catalyzed by O‐GlcNAc transferase and O‐GlcNAcase. O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc) is a nonselective inhibitor of O‐GlcNAcase, which increases the level of protein O‐GlcNAcylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3‐L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP‐binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3‐L1 adipocytes following treatment with PUGNAc. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 (E1), Hsp70, vasolin‐containing protein (Vcp), and Hsp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3‐L1 cells.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27‐hydroxycholesterol (27‐OH) and 24‐hydroxycholesterol (24‐OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK‐N‐BE human neuroblastoma cells with patho‐physiologically relevant amounts of 27‐OH and 24‐OH showed that both oxysterols induce a net synthesis of Aβ_1‐42 by up‐regulating expression levels of amyloid precursor protein and β‐secretase, as well as the β‐secretase activity. Interestingly, cell pretreatment with N‐acetyl‐cysteine (NAC) fully prevented the enhancement of β‐amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β‐amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols‐induced Aβ toxic peptide accumulation in the brain.     

Proteomics and PUGNAcity will overcome questioning of insulin resistance induction by nonselective inhibition of O‐GlcNAcase

PTMs are the ultimate elements that perfect the existence and the activity of proteins. Owing to PTM, not less than 500 millions biological activities arise from approximately 20 000 protein‐coding genes in human. Hundreds of PTM were characterized in living beings among which is a large variety of glycosylations. Many compounds have been developed to tentatively block each kind of glycosylation so as to study their biological functions but due to their complexity, many off‐target effects were reported. Insulin resistance exemplifies this problem. Several independent groups described that inhibiting the removal of O‐GlcNAc moieties using O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), a nonselective inhibitor of the nuclear and cytoplasmic O‐GlcNAcase, induced insulin resistance both in vivo and ex vivo. The development of potent and highly selective O‐GlcNAcase inhibitors called into question that elevated O‐GlcNAcylation levels are responsible for insulin resistance; these compounds not recapitulating the insulin‐desensitizing effect of PUGNAc. To tackle this intriguing problem, a South Korean group recently combined ATP‐affinity chromatography and gel‐assisted digestion to identify proteins, differentially expressed upon treatment of 3T3‐L1 adipocytes with PUGNAc, involved in protein turnover and insulin signaling.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Structural and functional studies of a trans‐acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α‐ and β‐ketoreduction

While the cis‐acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans‐acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35‐Å resolution. This KR naturally reduces both α‐ and β‐keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N‐acetylcysteamine‐bound β‐keto substrate to a D ‐β‐hydroxy product, but also an N‐acetylcysteamine‐bound α‐keto substrate to an L ‐α‐hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl‐phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α‐ketoreduction may not be extensive since a KR that naturally reduces a β‐keto group within a cis‐acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α‐keto substrate to generate the D ‐α‐hydroxy product. A sequence analysis of trans‐acyltransferase KRs reveals that a single residue, rather than a three‐residue motif found in cis‐acyltransferase KRs, is predictive of the orientation of the resulting β‐hydroxyl group. Proteins 2014; 82:2067–2077. © 2014 Wiley Periodicals, Inc.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the potential of boronic acids as inhibitors of OXA‐24/40 β‐lactamase

β‐lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β‐lactams is the expression of β‐lactamase enzymes. To overcome β‐lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β‐lactamases also contain the characteristic β‐lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non‐β‐lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem‐hydrolyzing class D β‐lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β‐lactamase inhibitors, nor are they effectively inhibited by the newest, non‐β‐lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β‐lactamases, and are not extensively characterized as inhibitors of class D β‐lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β‐lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA‐24/40. Several compounds were identified as effective inhibitors of OXA‐24/40, with K_i values as low as 5 μM. The X‐ray crystal structures of OXA‐24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β‐lactamases.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended‐spectrum‐β‐lactamases,AmpC β‐lactamases and co‐β‐lactamases in Enterobacteriaceae

A promising means of rapid screening of extended‐spectrum‐β‐lactamase (ESBL), AmpC β‐lactamase, and co‐production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β‐lactamase‐producing Enterobacteriaceae , including 15 ESBLs, 32 AmpCs, nine co‐producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co‐existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co‐β‐lactamase‐producing Enterobacteriaceae are endemic.