Design of a covalently bonded glycosphingolipid microarray

Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype “universal” GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G_M1, with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G_M2). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.     

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.     

Antimicrobial activity of carbon fiber fabric modified with a polymer-gentamicin complex

Modified and unmodified carbon fiber supports were treated with solutions of a polymer-gentamicin complex, possessing high antimicrobial activity and low toxicity. It was found that the antimicrobial activity of modified carbon fiber fabrics depended on the nature of the support and on the immobilization conditions. The highest antimicrobial activity was observed with phosphorus-containing carbon fiber ion exchanger in salt form.     

Near-infrared luminescent lanthanide (Er, Nd) complexes covalently bonded to a terpyridine-functionalized silica matrix.

New kinds of hybrid materials containing covalently bonded tris(dibenzoylmethanate)Ln complexes (Ln=Er, Nd) in a terpyridine-functionalized silica matrix have been prepared and their near-infrared luminescence properties reported.     

Liver metabolic reactions: tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation. II. N,N-dimethylaniline

Rat liver microsomal preparations enzymatically catalyze the N-demethylation and N-oxidation of dimethylaniline as well as the N-demethylation of dimethylaniline-N-oxide. Both compounds were used as substrates and the formation of formaldehyde and N-oxide were determined.Both demethylation and N-oxidation of dimethylaniline are dependent on NADPH. This cofactor also increases the demethylation of dimethylaniline-N-oxide, although it is not an absolute requirement. Nicotinamide increases the rate of formation of formaldehyde and N-oxide from dimethylaniline by a factor of about 4 and decreases the N-oxide demethylation by the same factor. The cofactor optimum consists of NADPH, nicotinamide, and magnesium ions for the demethylation and N-oxidation of dimethylaniline, and of NADPH alone for the demethylation of its N-oxide. The kinetic constants of the three test reactions have been determined under these optimal cofactor requirements.Various agents strongly influence the rates of product formation of the three test reactions studied. SH-blocking agents, the chelating agent EGTA, as well as nicotinamide influence the rates of formaldehyde formation from dimethylaniline and N-oxide demethylation in an opposite way. This demonstrates that, in the tertiary amine demethylation of dimethylaniline, a C-oxidation pathway is operative in addition to an N-oxidation pathway with subsequent N-oxide demethylation. The following influences on the actual metabolic reactions could be deduced from the effects of agents on the test reactions: SKF 525-A inhibits and phenobarbital pretreatment stimulates N-oxide demethylation; EDTA inhibits both the latter reaction and N-oxidation; EGTA and nicotinamide stimulate C-oxidation and inhibit N-oxide demethylation; SH-blocking agents inhibit C-oxidation and stimulate both N-oxidation and N-oxide demethylation.Quantitative and qualitative species differences with respect to cofactor requirement and effect of SKF 525-A have been observed between rat and pig liver microsomes. In addition, profound differences in subcellular localization and metabolic rates between dimethylaniline and other substrates are known. Thus it is unlikely that the three metabolic reactions dealt with in this report are characteristic of tertiarr amine N-dealkylation in general.     

Antimicrobial activity of the amine fraction of cucumber (Cucumis sativus) extract

Summary The amine fraction of an extract of 10-day-old cucumber seedlings was shown to have a growth inhibitory effect onStaphylococcus aureus andPseudomonas aeruginosa. This antimicrobial activity was associated with the presence of the polyamine spermidine in the extract.

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Resumen La fracción aminada de un extracto de plántulas de 10 días de edad mostró un efecto inhibidor sobre el crecimiento deStaphylococcus aureus yPseudomonas aeruginosa. Esta actividad microbiana estaba asociada con la presencia en el extracto de la poliamina espermidina.

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Résumé La fraction amine d'un extrait d'embryons de concombre de 10 jours d'âge s'est révélée posséder un effet d'inhibition de la croissance surStaphylococcus aureus et surPseudomonas aeruginosa. Cette activité anti-microbienne est associée avec la présence de la polyamine spermidine dans l'extrait.

     

RNA tertiary folding monitored by fluorescence of covalently attached pyrene

The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test system. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide (U107) for which derivatization at the 2'-position should not perturb the folded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4-P6, providing a site-specifically pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivatized P4-P6 with Mg(2+), the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg(2+) approximately Ca(2+) > Sr(2+)) correlated well with biochemical folding assays that involve nondenaturing gel electrophoresis. Furthermore, mutations in P4-P6 remote from the chromophore that shifted the Mg(2+) folding requirement on nondenaturing gels also affected in a predictable way the Mg(2+) requirement for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.     

Antioxidant activity of liver growth factor,a bilirubin covalently bound to albumin

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin–bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.     

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.     

Thermodynamic evaluation of a covalently bonded transition state analogue inhibitor: inhibition of beta-lactamases by phosphonates

Serine beta-lactamases are inhibited by phosphonate monoesters in a reaction that involves phosphonylation of the active site serine residue. This reaction is much more rapid than the hydrolysis of these inhibitors in solution under the same conditions. The beta-lactamase active site therefore must have the ability to stabilize not only the anionic tetrahedral transition states of the acyl transfer reactions of substrates but also the pentacoordinated transition state(s) of phosphyl transfer reactions. A series of p-nitrophenyl arylphosphonates have been synthesized and the rate constants for their inhibition of the class C beta-lactamase of Enterobacter cloacae P99 determined. There is no direct correlation between these rate constants and the dissociation constants of analogous aryl boronic acids, where the latter are believed to generate good tetrahedral transition state analogue structures. Thus, the mode of stabilization of pentacoordinated phosphorus transition states by the beta-lactamase active site is qualitatively different from that of tetrahedral transition states. Molecular modeling suggests that the difference arises from different positioning of the side chain and of one of the oxygen ligands. In principle, the quality of the stable tetrahedral phosphonate complex as a transition state analogue structure can be assessed from the effect of its formation on the stability of the protein. Phosphonylation of the P99 beta-lactamase, however, had little effect on the stability of the protein, as measured both by thermal and guanidine hydrochloride denaturation. Consideration of the results of similar experiments with the Staphylococcus aureus PC1 beta-lactamase, where considerable stabilization is observed in thermal melting and, to a lesser degree, in formation of the molten globule in guanidine hydrochloride, but not in the complete unfolding transition in guanidine, suggests that results from the method may be strongly influenced by the interactions of the ligand with its environment in the unfolded state of the protein. Thus, quantitative estimates of the quality of a covalently bonded transition state analogue cannot generally be achieved by this method.     

Effect of tertiary amine on the carbodiimide-mediated peptide synthesis

The effect of tertiary amine (DIEA) on reaction rate and product purity of a carbodiimide/HOBt-mediated peptide synthesis was studied. It was found that very rapid activation can be achieved using carbodiimide/HOBt in non-polar solvents, such as DCM. Although the HOBt is poorly soluble in DCM, the activation proceeds within 2 min, probably forming the HOBt-ester. By such a preactivation followed by a coupling in the presence of DIEA the rate of coupling is comparable with other rapid methods using BOP or TBTU, and no racemization was found in a model coupling (less than 0.1%). For comparison, syntheses of neurotensin by means of different coupling reagents (BOP, TBTU, OPfp-esters) and the DIEA-catalyzed coupling after carbodiimide/HOBt-activation under comparable conditions have shown that these procedures are of the same value in view of coupling efficiency and product purity.     

Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus.

A bacteriocin-producing Bacillus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.     

Carbodiimide-intermediated esterification of the inorganic phosphates and the effect of tertiary amine base.

Detailed analysis of appropriate 31P nuclear-magnetic-resonance spectra shows that under the usual laboratory conditions, carbodiimide-induced condensation of orthophosphoric acid in a number of solvents leads to condensation only slightly beyond the metaphosphate composition in the presence of strong tertiary amines; whereas in the absence of amine, the condensation proceeds into the ultraphosphate region about halfway between the metaphosphate and phosphoric anhydride compositions. With amine, the principal product consists of the cyclic trimetaphosphate anion, with one of the nonbridging oxygen atoms substituted by the urea resulting from hydration of the carboiimide, i.e., (O2-) P-O-P(O2-) -O-P(O) [N[CH(CH3)2] see article [C(O)NHCH(CH3)2]] for the condensation with diisopropylcarbodiimide. Without amine, the major product is the 1,5-mu-oxotetrametaphosphate anion see article. The well-known carbodiimide-mediated phosphorylation of alcohols with orthophosphoric acid is shown to be directly attributable to the high reactivity of the phosphate branch groups of the carbodiimide-generated ultraphosphates.     

The tertiary amine local anesthetic dibucaine binds to the membrane skeletal protein spectrin

The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene-based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near-UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy-driven and insensitive to an increase in the ionic strength of the buffer.