N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract

Abstract

There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.     

Production and chitinase-binding ability of lipo-chitopentaose nodulation factor

The penta-N-acetyl-chitopentaose 2 has been prepared by using recombinant E. coli strains harboring the nodC gene (encoding chitooligosaccharide synthase) from Azorhizobium caulinodans. Then, the deacetylase NodB removed the N-acetyl moiety from the nonreducing terminus of 2 to give tetra-N-acetyl-chitopentaose 3. N-Acylation of 3 with stearyl chloride was performed in DMF containing water and provided the corresponding lipo-chitopentaose nodulation factor 4. A binding chitinase assay indicated that 4 was much more stable than 3.     

Structural studies of heparitin sulfates

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain.The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.     

Glycosylation via mixed disulfide formation using glycosylthio-phthalimides and -succinimides as glycosylsulfenyl-transfer reagents

The silver salts of tetra-O-acetyl α- or -β-d-glycopyranosyl thiols 1a–4a react smoothly with N-bromophthalimide and N-bromosuccinimide to furnish glycosylthio-phthalimide (1b–4b) and -succinimide (1c–3c) derivatives. Reactions of these reagents with aliphatic, aromatic, and glycosyl thiols as well as with cysteine and glutathione result in the formation of glycosylated mixed disulfides under mild conditions and in good yields. The S-glycosyl-N-acylsulfenamides described here represent novel, convenient glycosylsulfenyl-transfer reagents in effecting glycosylation of various thiols, including sugars, amino acids and peptides, through disulfide formation and can, therefore, be useful in controlled glycosylation of proteins as well.     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

The effect of substitution of the <Emphasis Type="Italic">N</Emphasis>-acetyl groups of <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine residues in chondroitin sulfate on its degradation by chondroitinase ABC

Chondroitinase ABC is a lyase that degrades chondroitin sulfate, dermatan sulfate and hyaluronic acid into disaccharides. The purpose of this study was to determine the ability of chondroitinase ABC to degrade chondroitin sulfate in which the N-acetyl groups are substituted with different acyl groups. The bovine tracheal chondroitin sulfate A (bCSA) was N-deacetylated by hydrazinolysis, and the free amino groups derivatized into N-formyl, N-propionyl, N-butyryl, N-hexanoyl or N-benzoyl amides. Treatment of the N-acyl or N-benzoyl derivatives of bCSA with chondroitinase ABC and analysis of the products showed that the N-formyl, N-hexanoyl and N-benzoyl derivatives are completely resistant to the enzyme. In contrast, the N-propionyl or N-butyryl derivatives were degraded into disaccharides with slower kinetics compared to that of unmodified bCSA. The rate of degradation of bCSA derivatives by the enzyme was found to be in the order of N-acetyl>N-propionyl>>N-butyryl bCSA. These results have important implications for understanding the interaction of N-acetyl groups of glycosaminoglycans with chondroitinase ABC.     

Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Digestibility of Acetylated and Succinylated Proteins by Pepsin-Pancreatin and Some Intracellular Peptidases

The molecular sizes of hydrolysates of acetylated and succinylated caseins by pepsin-pancreatin were examined by gel filtration on Sephadex G-15. There were more large peptides (average residual number > 15) in the hydrolysates of the acylated caseins than there were in the hydrolysate of unmodified casein. In these large peptides from the acylated caseins the contents of N^ε-acyl-lysines were high. The digestibility of N^ε-acetyl and N^ε-succinyl lysine bonds in peptides by aminopeptidases [(EC 3.4.11.1) and (EC 3.4.11.2)] and watermelon carboxypeptidase [model enzyme of cathepsin A (EC 3.4.16.1)] was examined using digest of acylated caseins by pepsin, trypsin and α-chymotrypsin and some synthetic peptides. All peptidases released either N^ε-acetyl or N^ε-succinyl-lysine from peptides.

The hydrolytic processes of acetylated and succinylated proteins before and after intestinal absorption are discussed.     

Synthesis of novel selenium containing sulfa drugs and their antibacterial activities

Synthesis of 3-[4-(N-substituted sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyri-do[3′,2′:4,5]selenolo[3,2-d]pyrimidines,7-[4-(N-substituted sulfamoyl)phenyl]-7,8-dihydro-8-oxo-3,4-diphenylpyrimido[4′,5′:4,5]selenolo [2,3-c]pyridazines and 1-[4-(N-substituted sulfamoyl)phenyl]-1,11-dihydro 11-oxo-4-methylpyrimido[4′,5′:4,5]selenolo[2,3-b]quinolines is reported. 4-Amino-N-pyrimidine-2-ylbenzene sulfonamide (a), 4-amino-N-(2,6-dimethylpyrimidin-4-yl)benzene sulfonamide (b), N-[(4-aminophenyl)sulfonyl] acetamide (c) with N-ethoxymethyleneamino of selenolo pyridine, selenolo pyridazine and selenolo quinoline derivatives respectively were obtained starting from 1-amino-N ⁴-substituted sulfanilamides. Spectroscopic data (IR, ¹H NMR, ¹³C NMR and Mass spectral) confirmed the structure of the newly synthesized compounds. Substituted pyrimidines, pyridazines and quinolines were screened for antibacterial activity against gram-positive and gram-negative bacteria. Selenolo derivative of N-[(4-aminophenyl)sulfonyl] acetamide (substitutent of sulfacetamide c) showed strong bactericidal effect against all the tested organisms. Selenolo[3,2-d]pyrimidin (substitutent a) showed a good bactericidal effect against Serratia marcescens, Staphylococcus aureus and Escherichia coli. Compounds selenolo[2,3-c]pyridazine (substitutent b), selenolo[2,3-b]quinoline(substitutents c)) exhibited a moderate bactericidal effect against Serratia marcescens. None of the synthesized seleno pyridazines has a considerable antimicrobial activity against the tested organisms. The minimum inhibitory concentration (MIC) of the most active compound-3-[4-(N-acetyl sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyrido[3′,2′:4,5]selenolo [3,2-d]pyrimidine was 10 mg ml⁻¹.     

Reaction of Glycosylisothiocyanates with 2-Chloroethylamine. Synthesis and Structure of N-Nucleoside Analogues

Abstract

Per-O-acetyl-β--glycopyranosylisothiocyanates (3 and 4) were condensed with 2-chloroethylamine hydrochloride to afford N,N'-bis(per-O-acetyl–β--glycopyranosyl)-N-(2-thiazolin-2-yl)thioureas (5 and 6) through the glycosylaminoheterocycles (7 and 8) as intermediates. Compounds 7 and 8 were converted into N-(2-thiazolin-2-yl)urea or thioureas (9–11) by reaction with iso(thio)cyanates. Compounds 5, 6 and 9–11 show a strong chelated structure due to an intramolecular hydrogen bond, which anchors the E,Z conformation in solution.     

Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin

Two fungal strains were evaluated for β-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher β-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4–1) fractional factorial design of Response Surface Methodology. The high R² (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS–PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.     

The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

Viscosity and optical activity of chondroitin sulfate C

Chondroitin sulfate C (CSC), isolated from shark vertebral mucoprotein, has a molecular weight of 0.2 × 10⁴–5.5 × 10⁴ in both NaCl and CaCl₂ solutions. Optical rotatory dispersion and circular dichroism of CSC reveal a strong, negative, optically active band near 210 mμ, arising from the carboxylate and N-acetyl groups. Results of similar studies of glucuronic acid and N-acetyl-D -galactosamine suggest that the N-acetyl group contributes more to the rotations of CSC than does the carboxylate group, but the acidification of the carboxylate groups largely accounts for the change in magnitude and position of the circular dichroic bands of CSC at low pH.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

Thermochemistry of N-C bonds hydrolysis in amides,peptides, N-acetyl amino acids and high-energy N-C bonds hydrolysis in N-acetyl imidazole and urea

• 1.1. The reaction enthalpies of hydrolysis of amides, peptides and N-acetyl amino acids were calculated for both ionized and un-ionized forms of reaction components.
• 2.2. The average values of reaction enthalpies of amides, peptides and N-acetyl amino acids hydrolysis were essentially different from each other for ionized forms of reaction components and were equal for un-ionized forms of reaction components in the error interval.
• 3.3. As an example of high-energy N-C bonds N-acetyl imidazol and urea were discussed. It was found that the reaction enthalpies of hydrolysis of above compounds were different from analogous thermodynamic values of hydrolysis of amides, peptides and N-acetyl amino acids for any forms of components.

     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

Summary The preparation of the thymine peptide nucleic acid (PNA) monomer carrying a 2-nitrophenyl group in position 4 is described. This monomer is incorporated into PNA oligomers and reacted with amines to yield PNA oligomers carrying 5-methylcytosine derivatives. During the deprotection-modification step two side reactions were detected: degradation of PNA oligomer from theN-terminal residue and modification ofN ⁴-tert-butylbenzoyl cytosine residue. Protection of theN-terminal position and the use ofN ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

SYNTHESIS AND HYBRIDIZATION OF NOVEL CHIRAL PYRROLIDINE BASED PNA ANALOGUE

Four different PNA fragments containing units of either the R- or S- isomer of N-(2-pyrrolidine-methyl)-N-(thymine-1-acetyl)-glycine (Pmg) were synthesized on a solid support. UV thermal melting experiments with complementary RNAs were performed and it was found that R-Pmg containing PNAs bind better to RNA than those containing the S-Pmg units.     

Cell Surface Carbohydrate Differences in Wild and Mutant Strains of Crithidia fasciculata1

Wild type Crithidia fasciculata and three drug-resistant mutant strains that have shown “flagellar adherence” were studied as to their ability to agglutinate with lectins specific for receptor molecules containing N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose-like residues, fucose, and sialic acid. Escherichia coli with mannose-sensitive fimbriae was also used as an agglutination probe. The presence of D-GalNAc, D-Gal, and mannose-like residues was detected in the wild strain. Generally, in the mutants, residues of these sugar units were present in greater concentrations when compared to the wild type strain. β-Galactosidase treatment showed that β-D-Galp units are exposed on the cell membrane. All types of cell agglutination including flagellum-flagellum (F-F), flagellum-soma (F-S), and soma-soma (S-S) were observed when lectins were used; however, with E. coli only the F-F type of cell agglutination was observed with the wild type strain and the TFR^R1 mutant. All types of agglutination were observed with the other two mutants.