E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.     

N-2′-Acetoxybenzoyl derivatives of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose

N-2′-Acetoxybenzoyl (aspirin) derivatives (degree of substitution 0·35–1·00) of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose were prepared by methods that gave yields in the range 65–86%. The salicylate of chitosan was isolated with a 98% yeild. Aspirin or salicylic acid was released much more slowly from N-(2′-acetoxybenzoyl)-chitosan than from the salicylate of chitosan, and much faster at 37°C in 0·1 m NaOH solution than in 2% aqueous acetic acid solution. Salicylic acid was isolated from the dialysate (0·1 m NaOH solution) of N-(2′-acetoxybenzoyl)-chitosan.     

The effect of hydrogen bonding in two crystal modifications of aquabis(N,N-dimethylglycinato-κN,κO)copper(II): Experimental and theoretical study

From the crystals of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) dihydrate (compound 1, space group P2₁2₁2₁) novel crystal structure of trans aquabis(N,N-dimethylglycinato-κN,κO)copper(II) (compound 2, space group Pbca) was obtained and analysed by X-ray diffraction. In the crystal structure 1, the O-H?O hydrogen bonds form three-dimensional network. In the crystal structure 2, two-dimensional layers stacking to each other are formed, with non-polar N,N-dimethyl groups placed on the opposite sides of the layers, and with the polar part in the middle forming CO?O-H and C-H?O hydrogen bonds. Different hydrogen bonding patterns in 1 and 2 do not pronouncedly affect molecular geometry of the title compound. Molecular mechanics force field suited for studying the properties of bis(amino acidato)copper(II) complexes in the solid state can follow the differences between the experimental molecular structures in the two diverse crystalline surroundings. To make possible direct comparison between crystal lattices, the force field was applied to predict unit cell packing of supposed anhydrous bis(N,N-dimethylglycinato)copper(II) in space group Pbca. Relative intermolecular energies of hypothetic anhydrous crystal and simulated 1 and 2 crystals are discussed. On the basis of experimental and theoretical results we conclude that the main effect of two water molecules of crystallisation in 1 is to stabilise the crystal packing via hydrogen bonding, whilst similar pyramidal copper(II) coordination geometry in 1 and 2 is due to axially coordinated water molecule and its intermolecular interactions.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked (N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

A homologous series of apoptosis-inducing N‑acylserinols: Thermotropic phase behavior,interaction with cholesterol and characterization of cationic N‑myristoylserinol-cholesterol-CTAB niosomes

N?Acylserinols (NASOHs) exhibit anti-cancer activity by elevating ceramide levels, and/or by activating proapoptotic effectors. In the present work we investigated the thermotropic phase behavior and supramolecular organization of a homologous series of NASOHs (number of C-atoms in the acyl chain, n?=?8–18), and the interaction of N-myristoylserinol (NMSOH) with cholesterol, and characterized cationic niosomes made up of NMSOH, cholesterol and cetyltrimethylammonium bromide (CTAB). Differential scanning calorimetric studies revealed that NASOHs exhibit a major chain-melting phase transition in both dry and hydrated states. The thermodynamic parameters, transition enthalpy and entropy show linear dependence on the acyl chain length in the dry state, but exhibit odd-even alternation in the hydrated state. Powder X-ray diffraction studies revealed that NASOHs adopt a tilted bilayer structure, wherein the bilayer repeat distances (d-spacings) also showed odd-even alteration, with even-chainlength compounds exhibiting slightly higher d-spacings. Studies on the interaction between NMSOH and cholesterol revealed that both lipids mix well with up to 55?mol% cholesterol, whereas phase separation was observed at higher cholesterol content. The transition enthalpy corresponding to the NMSOH-cholesterol complex increases up to 55?mol% cholesterol and decreases at higher cholesterol content. Presence of the cationic surfactant CTAB affects the phase behavior, fluidity and size of the NMSOH-cholesterol (45,55, mol/mol) niosomes, with unilamellar vesicles of about 85 (±20) nm in diameter being obtained at 10?mol% CTAB. These results provide a thermodynamic and structural basis for further investigations on these cationic niosomes towards their use in drug delivery applications, especially for anticancer drugs.     

Antithrombin conformation and the catalytic role of heparin. I. Does cleavage by thrombin induce structural changes in the heparin-binding region of antithrombin?

Heparin has been shown to exhibit lower affinity for the antithrombin-thrombin complex than for antithrombin alone (Carlstrom, A.-S., Lieden, K., and Bjork, I. (1977) Thromb. Res. 11, 785-797), suggesting that structural alterations in antithrombin may accompany its reaction with thrombin. The hydroxy-nitrobenzyl (HNB) group attached to a unique tryptophan has been used in the present study as an extrinsic probe for localization of conformational changes to the heparin-binding region within antithrombin III using immunochemical and spectral techniques. Site-specific modification of tryptophan-49 in antithrombin with the hydroxynitrobenzyl reagent blocks heparin binding to the protein and provides a chemical label in the heparin-binding region of the protein (Blackburn, M. N., Smith, R. L., Carson, J., and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941). Antibodies specific for the hydroxynitrobenzyl hapten, which bind to HNB-tryptophan-49 in antithrombin, were used to detect a change in conformation in the region of tryptophan-49 which occurs upon thrombin binding to antithrombin. This thrombin-induced structural change was also apparent from spectral perturbations which were detected with the environmentally sensitive HNB moiety. Thus, the HNB group was used as an immunochemical probe as well as a spectral reporter group to provide insight into an allosteric mechanism of control in the catalytic role of heparin. The thrombin-promoted alteration of the structure in the heparin-binding region is presumably responsible for recycling of heparin, allowing it to catalyze further reactions between antithrombin and thrombin.     

Characterization of the multiple forms of β-xylanase produced by a Streptomyces sp. growing on lignocellulose

Summary Streptomyces sp. strain EC10 degraded efficiently the hemicellulose fraction of wheat straw. Three forms of -xylanases detected in the culture filtrate were purified by precipipation with ammonium sulphate, chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-100. The three purified enzymes (X _ia , X _ib and X _ii ) were homogeneous by polyacrylamide gel electrophoresis. The enzymes were typical non-debranching endo--xylanases (1,4--d-xyla xylanohydrolases; E.C.3.2.1.8) with respective relative molecular weights of 32,000, 22,000 and 21,000 and isoelectric points of 6.8, 8.9 and 5.2. The enzymes were highly specific for xylans and showed optimal activity at pH 7.0–8.0 and 60°C. The preparations were completely free from cellulolytic activity (endoglucanase) and showed high thermal stability. No synergy between the three enzymes was detected for complete xylan hydrolysis of deacetylated arabino- and glucuronoxylans.Offprint requests:to: M. J. Penninckx     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Determination of sequence and linkage of tissue oligosaccharides in caprine β-mannosidosis by fast atom bombardment,collisionally activated dissociation tandem mass spectrometry

Fast atom bombardment, collisionally activated dissociation tandem mass spectrometry (FAB-CAD-MS/MS), combined withp-aminobenzoic acid ethyl ester (ABEE) derivatization, were used to confirm the sequence and linkage pattern of subnanomolar amounts of the previously characterized three major thyroid gland oligosaccharides accumulated in caprine -mannosidosis. Positive ion FAB-CAD-MS/MS of both the [M + H]⁺ and [M + Na]⁺ ions from the ABEE derivatized oligosaccharides produced product ions derived from cleavage of the glycosidic bonds which allowed the sequences to be determined. Several fragments resulting from cleavages across the sugar ring permitted the assignment, in some cases, of the linkage positions between the sugar residues. The natriated molecule yielded several fragments of this type which were not observed when the protonated molecule was selected as the precursor ion. Use of these techniques gave the complete sequence and linkage characterization of the disaccharide and complete sequence and partial linkage information for the two higher oligosaccharides.     

Modeling peptide formation during the hydrolysis of β-casein by Lactococcus lactis

Hydrolysis of milk proteins by lactic acid bacteria leads to the formation of a large number of peptides. In this work, the hydrolysis of β-casein by the protease PrtP_I of Lactococcus lactis was studied. Experiments were carried out at different initial enzyme/substrate ratios. Identification and quantification of peptides were performed by MS and RP-UHPLC analyses. Nine low molecular weight (LMW) peptides were quantified absolutely. Additionally, semi-quantification of six high molecular weight peptides (HMW) was provided. To describe the dynamics of peptides concentrations, an aggregated model was developed. This model links peptide formation to the breakdown of intact protein by introducing the concept of virtual intermediate peptides (VIP). The model represented the experimental data with an average error of 14% (comparable with the experimental error). By using the model, three dynamic pools of peptides were identified. The model suggests that LMW peptides have similar dynamic characteristics as their counterpart HMW peptides in the β-casein sequence. This study indicates that the presence and structure of micelles affect the hydrolysis dynamics and that, for some peptides, the enzyme/substrate ratio appears to affect the hydrolysis stoichiometry. The model developed is parsimonious and has a basic mechanistic component. It allows for a rational study of protein hydrolysis.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

Monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by protease in N,N-dimethylformamide with low water content

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) laurate was synthesized from AA-2G and vinyl laurate with a protease from Bacillus subtilis in N,N-dimethylformamide (DMF) with low water content. Addition of water to DMF dramatically enhanced monoacyl AA-2G synthesis. Maximum synthetic activity was observed when 3% (v/v) water was added to the reaction medium. Under the optimal reaction conditions, 5-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid, 2-O-(6′-O-dodecanoyl-α-d-glucopyranosyl)-l-ascorbic acid, and 6-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid were synthesized in yields of 5.5%, 3.2%, and 20.4%, respectively.     

Change of the Structure of Cell Wall β-l,3-d-Glucan with the Growth of Neurospora crassa Cells

The chemical structure of cell wall β-d-glucans as well as the activities of lytic enzymes such as β-1,3-d-glucanase and β-1,6-d-glucanase changed during the growth of Neurospora crassa.

A dramatic change in the cell wall β-d-glucan structure was observed between cells of the middle logarithmic phase and ones of the late logarithmic phase. The ratio of 1,3-linked glucose residues to non reducing terminal glucose residues decreased from 85 to 55 and the ratio of gentiobiose as a hydrolysis product with exo-β-1,3-d-glucanase increased significantly between the two phases.

Two prominent peaks of β-1,3-d-glucanase as well as the β-1,6-d-glucanase activities appeared in the culture filtrate at different growth stages, the early logarithmic phase and the stationary phase. In the cell wall, β-d-glucosidase activity instead of the β-l,6-d-glucanase and β-1,3-d-glucanase activities was observed in the late logarithmic phase.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

Wnt5b stimulates adipogenesis by activating PPARγ, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a

Correct Wnt signaling is required for adipogenesis and alterations occur in Type 2 diabetes mellitus (T2DM). Gene expression studies showed that β-catenin independent Wnt5b was down-regulated in T2DM preadipocytes, while its paralog Wnt5a was unchanged. Our study aimed at defining the expression profile and function of Wnt5a and Wnt5b during adipogenesis by determining their effect on aP2 and PPARγ expression and assessing the level of β-catenin translocation in mouse 3T3-L1 preadipocytes. Additionally, we explored the effect on adipogenic capacity by Wnt5b overexpression in combination with stimulation of the β-catenin dependent or β-catenin independent Wnt signaling. Expression of Wnt5b was, like Wnt5a, down-regulated upon induction of differentiation and both inhibit β-catenin dependent Wnt signaling at the initiation of adipogenesis. Wnt5b additionally appears to be a potent enhancer of adipogenic capacity by stimulation of PPARγ and aP2. Down-regulation of Wnt5b could therefore contribute to decreased adipogenesis observed in T2DM diabetic subjects.     

Synthesis, X-ray crystal structure and magnetic study of the 1D {[Cu2(N,N,N′,N′-tetramethyl-ethylenediamine)2(μ-1,5-dca)2(dca)2]}n complex

Reaction of Cu(NO₃)₂ · 3H₂O, N,N,N′,N′-tetramethyl-ethylenediamine (L) and sodium dicyanamide (Nadca) in aqueous medium yields a complex the {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n complex, 1. Single crystal X-ray analysis reveals that complex 1 has a 1D infinite chain structure in which copper(II) ions are bridged by single dicyanamide anions in an end-to-end fashion. The coordination environment around copper(II) is distorted square pyramidal. Two among the four coordination sites of the basal plane are occupied by the nitrogen atoms of the diamine and two remaining sites are occupied by the terminal nitrogen atom of a bridging and of a monodentate dca anions. The fifth coordination site (apical) is occupied by a nitrogen atom from a bridging dca anion of an adjacent CuL(dca)₂ moiety, yielding the [Cu₂L₂(μ-1,5-dca)₂(dca)₂] dinuclear unit. Dimeric units are connected to each other by single μ-1,5-dicyanamido group to form infinite 1D chains which propagate parallel to the crystallographic c-axis. The variable temperature magnetic susceptibility measurements evidenced weakly antiferromagnetic interactions (J = −0.26 cm⁻¹) in {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n, 1.     

SMN, Gemin2 and Gemin3 Associate with β-Actin mRNA in the Cytoplasm of Neuronal Cells In Vitro

Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of β-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that β-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.