Structural Influence of Sugar and Aglycon Moieties on the Acetolysis of Some Glycosylamines

The acetolysis was applied to a novel cleavage of N-p-tolyl-β-d-glycosylamines in glucogalacto- and manno-configurations and their 2-acetamido-2-deoxy sugars. The results were compared with the acetolysis of furanosyl- and pyranosylnucleosides. N-Acetyl-p-toluidine was isolated as crystals in up to 92% yield in the acetolysis of N-P-tolyl-β-d-glycosylamines of neutral sugars and in 1.5~2.0% yields in that of 2-acetamido-2-deoxy sugars. The stability of glycosylamine linkages against the acetolysis was found in the following order: pyranosylnucleosides (the most stable) > N-P-tolyl-β-d-glycosylamine of 2-acetamido-2-deoxy sugars > furanosylnucleosides > N-P-tolyl-β-d-glycosylamines of neutral sugars (the most unstable).     

Interaction ofN-acetyl-4-, 7-, 8- or 9-deoxyneuraminic acids andN-acetyl-4-, 7- or 8-mono-epi- and-7,8-di-epineuraminic acids withN-acetylneuraminate lyase

Various deoxy- and epi-derivatives ofN-acetylneuraminic acid were synthesized and tested for their substrate properties withN-acetylneuraminate lyase fromClostridium perfringens.N-Acetyl-9-deoxyneuraminic acid is a good substrate,N-acetylneuraminic acid derivatives with epimeric configuration at C-7, C-8 or both are cleaved slowly, whileN-acetyl-4-epi-,N-acetyl-4-deoxy-,N-acetyl-7-deoxy-andN-acetyl-8-deoxyneuraminic acid are resistant to enzyme action.N-Acetyl-4-deoxyneuraminic acid andN-acetyl-4-epineuraminic acid competitively inhibit the enzyme. These studies give further insight into a mechanism proposed for the reversible cleavage of sialic acids byN-acetylneuraminate lyase.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

The effect of varying nitrogen and phosphorus availability on nutrient use by Larrea tridentata,a desert evergreen shrub

Summary In a phytotron study of the effects of nitrogen and phosphorus supply ratio on nutrient uptake and use by Larrea tridentata, seedlings responded to increases in N and P availability with increases in leaf size, total biomass, and leaf nutrient concentration, and with decreases in root: shoot ratio. N and P use efficiency decreased with increasing N and P availability, respectively, but increased with increasing availability of the other nutrient, suggesting that Larrea responds both to the absolute and to the relative availability of limiting nutrients. Absolute amounts of N and P resorption, as well as N and P resorption efficiencies did not demonstrate a significant trend with nutrient availability, and there was no evidence of significant interactions between the two nutrients. More studies of the effects of nutrient interactions in the cycling and use of nutrients by different plant species are needed before more general conclusions can be drawn.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.     

Metabolism of <Emphasis Type="Italic">N</Emphasis>-Methyl, <Emphasis Type="Italic">N</Emphasis>-propargylphenylethylamine: Studies with Human Liver Microsomes and cDNA Expressed Cytochrome P450 (CYP) Enzymes

Summary 1. We used an in vitro screening procedure and studies with individual human liver microsomes and cDNA-expressed CYP enzymes to investigate the metabolism of the putative neuroprotective drug N-methyl,N-propargyl-2-phenylethylamine (MPPE) to N-methylphenylethylamine (N-methylPEA) and N-propargylphenylethylamine (N-propargylPEA). 2. An electron-capture gas chromatographic procedure previously developed in our laboratories was used to measure the quantities of N-methylPEA and N-propargylPEA formed in the experiments with a single donor human liver microsome panel and cDNA expressed single CYP enzyme systems. The data were fitted to nonlinear regressions using Prism to determine kinetic constants. The results from a fluorogenic screen determined which cDNA-expressed single CYP enzymes were investigated. 3. CYP2B6, CYP2C19, and CYP2D6 all contributed to the formation of N-methylPEA, while only CYP2B6 catalyzed the formation of N-propargylPEA. The K _M and V _max values for N-propargylPEA formation were 290 ± 70 μM and 139±16 ng/mL/min. The values for formation of N-methylPEA were not determined from these experiments due to the complexity of fitting the data to a three-variable equation, but data on the time course of N-methylPEA formation are presented. 4. Catabolism of MPPE to N-methylPEA and N-propargylPEA is catalyzed by CYP enzymes. CYP2B6, 2C19 and 2D6 all contribute to the depropargylation of the parent compound, but only CYP2B6 also catalyzes demethylation. CYP2C19 was found to be the most active with respect to generation of N-methylPEA.     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

东方蜜蜂微孢子虫N6-腺苷特异性甲基化转移酶基因的克隆、分子特性及表达模式北大核心

【目的】东方蜜蜂微孢子虫(Nosme ceranae)专性侵染成年蜜蜂导致微孢子虫病,给养蜂生产造成很大损失。目前,东方蜜蜂微孢子虫的N6-腺苷特异性甲基化转移酶(N6-adenine-specific methyltransferase,N6AMT)基因NcN6AMT的研究仍然缺失。本研究对NcN6AMT的编码序列(coding sequence,CDS)区进行克隆,并解析NcN6AMT蛋白的理化性质和分子特性,进而测定东方蜜蜂微孢子虫侵染意大利蜜蜂(Apis mellifera ligustica)和中华蜜蜂(Apis cerana cerana)工蜂过程中NcN6AMT的相对表达量,以期丰富NcN6AMT的信息,并为探究东方蜜蜂微孢子虫侵染过程NcN6AMT的功能及表观调控机制提供基础。【方法】采用Protparam和ProtScale软件对NcN6AMT进行等电点和亲水性分析。通过SignalP 5.0、NetPhos 3.1、TMHMM-2.0、SOPMA和SWISS-MODEL等软件分别预测NcN6AMT的信号肽、磷酸化位点、跨膜结构域、二级结构和三级结构。使用WoLF PSORT II软件预测NcN6AMT的亚细胞定位。根据N6AMT氨基酸序列,通过TBtools软件对智人(Homo sapiens)、小鼠(Mus musculus)、褐飞虱(Nilaparvata lugens)、兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠脑炎微孢子虫(Encephalitozoon intestinalis ATCC 50506)、蚱蜢脑炎微孢子虫(Encephalitozoon romaleae SJ-2008)、美洲思普雷格孢虫(Spraguea lophii 42_110)、家蚕微孢子虫(Nosema bombycis CQ1)、隐生菱形藻(Nitzschia inconspicua)和东方蜜蜂微孢子虫(Nosema ceranae)的N6AMT进行结构域预测和分析。利用MEME软件和MEGA 11.0软件进行东方蜜蜂微孢子虫和其他物种N6AMT的保守基序预测及进化树构建。采用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)检测NcN6AMT在东方蜜蜂微孢子虫侵染意大利蜜蜂和中华蜜蜂工蜂过程的相对表达量。【结果】通过PCR扩增出大小约500 bp的目的片段,克隆测序结果显示其与GenBank数据库收录的预测序列一致;NcN6AMT蛋白的分子量约为18.7 kDa,分子式为C845H1374N214O249S6,理论等电点为5.88,脂溶系数是119.76,不稳定系数为37.47,平均亲水系数为0.025,含166个氨基酸和15个磷酸化位点,不含典型的跨膜结构域和信号肽,可同时定位于细胞质、线粒体、细胞核和液泡膜;NcN6AMT含1个甲基转移酶小结构域(methyltransferase small domain,MTS),该结构域同样存在于家蚕微孢子虫和兔脑炎微孢子虫等8个其他物种的N6AMT;在东方蜜蜂微孢子虫、兔脑炎微孢子虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的N6AMT中均预测到5个相同的保守基序;NcN6AMT与家蚕微孢子虫、肠脑炎微孢子虫、兔脑炎微孢子虫和蚱蜢脑炎微孢子虫的N6AMT序列一致性达到70.92%;东方蜜蜂微孢子虫和家蚕微孢子虫的N6AMT在系统进化树上聚为一支;东方蜜蜂微孢子虫接种后1–4 d,NcN6AMT在意大利蜜蜂和中华蜜蜂工蜂中肠内均呈现先上升后下降的表达趋势。【结论】成功克隆到NcN6AMT基因的CDS区,明确了NcN6AMT蛋白的理化性质和分子特性,并揭示东方蜜蜂微孢子虫和家蚕微孢子虫的N6AMT蛋白具有较高的保守性,NcN6AMT在东方蜜蜂微孢子虫侵染意大利蜜蜂和中华蜜蜂工蜂的第一个增殖周期(1–4 dpi)内动态表达且均呈上升-下降的表达模式。     

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5′-diphospho-N-[²H]-acetyl-glucosamine and the chemical preparation of N-[²H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.     

Purification,properties and primary structure of H2-formingN 5,N10-methylenetetrahydromethanopterin dehydrogenase fromMethanococcus thermolithotrophicus

H₂-FormingN ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized fromMethanobacterium thermoautotrophicum and fromMethanopyrus kandleri. We report here on the purification and properties of the enzyme fromMethanococcus thermolithotrophicus. Thehmd gene was cloned and sequenced. The results indicate that the enzyme fromMc. thermolithotrophicus is functionally and structurally closely related to the H₂-forming methylene tetrahydromethanopterin dehydrogenase fromMb. thermoautotrophicum andMp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.Abbreviations H ₂ Forming dehydrogenase orHmd - H₂-FormingN ⁵,N¹⁰ methylene tetrahydromethanopterin dehydrogenase - H ₄MPT Tetrahydromethanopterin - CH ₂=H₄MPT N⁵,N¹⁰ Methylene tetrahydromethanopterin - CHH ₄MPT⁺ N⁵,N¹⁰ Methenyltetrahydromethanopterin - MALDI-TOF-MS Matrix-assisted laser desorption     

Photosynthesis,Transpiration, and Water Use Efficiency of Vegetative and Reproductive Shoots of Grassland Species from North-Eastern China

The differences in net photosynthetic rate (P _N), transpiration rate (E), and water use efficiency (WUE) between the vegetative and reproductive shoots of three native grass species from the grassland of northeastern China [grey-green and yellow green populations of Leymus chinensis (Trin.) Tzvel., Puccinellia tenuiflora (Griseb) Scrib & Merr, Puccinellia chinampoensis Ohwi] were compared. The two type shoots experienced similar habitats, but differed in leaf life-span and leaf area. The leaf P _N and WUE for the vegetative shoots were significantly higher than those for the reproductive shoots in the grasses, while their E were remarked lower in the dry season. Relative lower leaf P _N and WUE for the reproductive shoots of grassland grasses may explain the facts of lower seed production and the subordinate role of seed in the grassland renewal in north-eastern China.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.     

[N,N′-Bis(salicylidene)-1,2-phenylenediamine]metal complexes with cell death promoting properties

We developed N,N′-bis(salicylidene)-1,2-phenylenediamine (salophene, 1) as a chelating agent for metal ions such as Mn(II/III), Fe(II/III), Co(II), Ni(II), Cu(II), and Zn(II). The resulting complexes, from which owing to the carrier ligand a selective mode of action is assumed, were tested for antiproliferative effects on the MCF-7 breast cancer cell line. The cytotoxicity in this assay depended on the nature of the transition metal used. Iron complexes in oxidation states +II and +III (3, 4) strongly reduced cell proliferation in a concentration-dependent manner, whereas, e.g., the manganese analogues 5 and 6 were only marginally active. Therefore, the [N,N′-bis(salicylidene)-1,2-phenylenediamine]iron(II/III) complexes 3 and 4 were selected for studies on the mode of action. Both complexes possessed high activity against various tumor cells, for instance, MDA-MB-231 mammary carcinoma cells as well as HT-29 colon carcinoma cells. They were able to generate reactive oxygen species, showed DNA binding, and induced apoptosis. Exchange of 1 by N,N′-bis(salicylidene)-1,2-cyclohexanediamine (saldach, 2) yielding complexes 7 and 8 reduced the in vitro effects drastically. An unequivocal mode of action cannot be deduced from these results, but it seems to be very likely that cell death is caused by interference with more than one intracellular target. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.