Purification and characterization of a nuclease from Lentinus edodes.

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.     

Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid

The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by successive gamma-deoxygenation through the cyclocarbonates, separation from the (alphaS,betaR)-isomer by means of flash chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNA(Phe) was isolated on a scale of 100 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P(1), followed by reverse-phase column chromatography, complete digestion with nuclease P(1)/alkaline phosphatase, and reverse-phase HPLC. Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (alphaS,betaS)-11.     

Practical synthesis of wybutosine, the hypermodified nucleoside of yeast phenylalanine transfer ribonucleic acid

An improved synthesis of 3-beta-D-ribofuranosylwybutine (2) has been achieved by the Wittig reaction between 4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]-purine-7-carbaldehyde (8) and the phosphorane derived from (R)-2-[(methoxycarbonyl)amino]-3-(triphenylphosphonio)propanoate (9), followed successively by methylation, hydrogenation, and deprotection. On the other hand, the minor nucleoside wybutosine of yeast tRNA(Phe) was isolated on a scale of 80 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P1, followed successively by reversed-phase column chromatography, complete digestion with nuclease P1/alkaline phosphatase, and reversed-phase HPLC. Comparison of this nucleoside with 2 has unambiguously established that the structure of wybutosine is (alphaS)-alpha-[(methoxycarbonyl)-amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester (2).     

Electrophoretic extraction and analysis of DNA from chitosan or poly-l-lysine-coated alginate beads

Alginate beads containing entrapped DNA were produced using both external and internal calcium sources, and coated with chitosan or poly-l-lysine membranes. The beads were assayed with DNase nuclease to determine formulation conditions offering the highest level of DNA protection fromnucleic acid hydrolysis, simulating gastrointestinal exposure. A method was developed to extract and assay intracapsular DNA through a modified agarose electrophoresis system. Both external and internally gelled beads were permeable to DNase (M_w=31 kDa), indicated by the absence of DNA after nuclease exposure. At low levels of DNase exposure, coated high guluronic content alginate beads offered a higher level of DNA protection compared with coated beads with low guluronic alginate. No apparent correlation was found with chitosan membrane molecular weight and degree of deacetylation; however, increasing poly-l-lysine molecular weight appeared to increase DNase exclusion from beads. At elevated levels of DNase exposure, DNA hydrolysis was evident within all coated beads with the exception of those coated with the highest molecular weight poly-l-lysine (M_w=197.1 kDa), which provided almost total nuclease protection. Optimal combination then for DNA protection from nucleases is a high guluronic alginate core, coated with high molecular weight poly-l-lysine.     

New Thermostable Amylase from <Emphasis Type="Italic">Bacillus cohnii</Emphasis> US147 with a Broad pH Applicability

A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed K _m and V _max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO₃, 1 mM H₂O_2, 1 mM Zn⁺², and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca ²⁺ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.     

Zn(2+) Responsive Bimodal Magnetic Resonance Imaging and Fluorescent Imaging Probe Based on a Gadolinium(III) Complex

A Zn(2+)-responsive bimodal magnetic resonance imaging (MRI) and luminescence imaging probe GdL was synthesized. The relaxivity and luminescence properties were examined. In the presence of 0.5 equiv of Zn(2+), the longitudinal relaxivity is increased from 3.8 mM(-1) s(-1) to 5.9 mM(-1) s(-1) at 23 MHz and 25 °C with 55% enhancement, whereas the fluorescence exhibits a 7-fold increase. The Zn(2+) responsive imaging probe shows favorable selectivity and tolerance over a variety of biologically relevant anions and metal ions in physiological pH range for both relaxivity and luminescence. In vitro phantom images and confocal fluorescence images in living cells show that the bimodal Zn(2+) probe can effectively enhance T(1)-weighted imaging contrast and luminescence imaging effect through Zn(2+) coordination with excellent cellmembrane permeability and biocompatibility. Spectral and electrospray ionization mass spectrometry (ESI-MS) studies indicate that two different Zn(2+)-bound species, (GdL)(2)Zn and GdLZn, are formed when 0.5 and 1 equiv of Zn(2+) are bound to GdL complex, respectively. Crystal structural determination and dysprosium-induced (17)O NMR shift (DIS) experiment demonstrate that the increased molecular weight and the improved molecular rigidity upon complexation of Zn(2+) with GdL is the primary factor for relaxivity enhancement. Significant enhancement of the luminescence is due to a heavy atom effect and much increased molecular rigidity upon Zn(2+) binding to 8-sulfonamidoquinoline chromophore.     

In vitro determination of the release kinetics of peptides and free amino acids during the digestion of food proteins

The kinetics of peptide release during in vitro digestion of 4 protein sources (casein, cod protein, soy protein, and gluten) were investigated. Samples were sequentially hydrolyzed with pepsin (30 min) and pancreatin (2, 4, or 6 h) in a dialysis cell with continuous removal of digestion products. Nondialyzed digests were fractionated by ion-exchange chromatography and ultrafiltration. Animal proteins were digested at a greater rate than plant proteins. Target amino acids of specific enzymes appeared more rapidly in the dialyzed fractions when compared to other amino acids. Throughout the hydrolysis, nondialyzed digests contained a higher proportion of peptide mixtures with basic-neutral properties. Except for gluten, peptide fractions with molecular weights that exceeded 10 kDa (basic-neutral, BN > 10) were rapidly hydrolyzed during the first 2 h of pancreatin digestion. The kinetics of release and the composition of peptide fractions were different when the protein sources were compared. The analysis of amino acids revealed that threonine and proline proportions were relatively high in BN > 10 and in peptide fractions with molecular weight between 10-1 kDa (BN 10-1), while tyrosine, phenylalanine, lysine, and arginine predominated in the low molecular weight (<1 kDa) fractions. More resistant peptides were generally rich in proline and glutamic acid. The role of in vitro digestion assays in dietary protein quality evaluation is discussed.     

A three-point method for evaluations of AMBER force field parameters: an application to copper-based artificial nucleases

We present the theoretical evaluation of new AMBER force field parameters for 12 copper-based nucleases with bis(2-pyridylmethyl) amine, 2,2′-dipyridylamine, imidazole, N,N-bis(2-benzimidazolylmethyl) amine and their derivative ligands based on first-principles electronic structure calculations at the B3LYP level of theory. A three-point approach was developed to accurately and efficiently evaluate the force field parameters for the copper-based nucleases with the ligands. The protocol of RESP atomic charges has been used to calculate the atomic charge distributions of the studied copper-based nucleases. The evaluated force field parameters and RESP atomic charges have been successfully applied in the testing molecular mechanics calculations and molecular dynamics simulations on the nucleases and the nuclease–DNA complexes, respectively. It has been demonstrated that the developed force field parameters and atomic charges can consistently reproduce molecular geometries and conformations in the available X-ray crystal structures and can reasonably predict the interaction properties of the nucleases with DNA. The developed force field parameters in this work provide an extension of the AMBER force field for its application to computational modeling and simulations of the copper-based artificial nucleases associated with DNA.     

Microwave assisted extraction for trace element analysis of plant materials by ICP-AES

Application of microwave assisted extraction for the decomposition and dissolution of plant samples for trace metal determination by ICP-AES was examined. Dried onion, leaves of spinach beet and three reference materials CTA-OTL-1, CTA-VTL-2 and CL-1 were analyzed. Water, EDTA and hydrochloric acid (0.01, 0.10 and 1.0 M, respectively) were used as leaching solutions. The extraction efficiency was investigated by comparison of the results with those obtained after microwave wet digestion. HCl was found to be very suitable for quantitative extraction of B, Ba, Cd, Cu, Mn, Ni, Pb, Sr and Zn from the samples. For reference materials, the measured concentrations are well consistent with the certified values. The use of EDTA led to a complete extraction of B, Cd, Ni, Pb, Sr and Zn. Water was found to be a good leaching solution for boron. For extraction with HCl and EDTA, the RSD values for the concentrations measured were below 8% for most of the elements.     

Selective covalent conjugation of phosphorothioate DNA oligonucleotides with streptavidin

Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.     

Microwave assisted extraction for trace element analysis of plant materials by ICP-AES

Application of microwave assisted extraction for the decomposition and dissolution of plant samples for trace metal determination by ICP-AES was examined. Dried onion, leaves of spinach beet and three reference materials CTA-OTL-1, CTA-VTL-2 and CL-1 were analyzed. Water, EDTA and hydrochloric acid (0.01, 0.10 and 1.0 M, respectively) were used as leaching solutions. The extraction efficiency was investigated by comparison of the results with those obtained after microwave wet digestion. HCl was found to be very suitable for quantitative extraction of B, Ba, Cd, Cu, Mn, Ni, Pb, Sr and Zn from the samples. For reference materials, the measured concentrations are well consistent with the certified values. The use of EDTA led to a complete extraction of B, Cd, Ni, Pb, Sr and Zn. Water was found to be a good leaching solution for boron. For extraction with HCl and EDTA, the RSD values for the concentrations measured were below 8% for most of the elements. Received: 4 May 2000 / Revised: 20 June 2000 / Accepted: 22 June 2000     

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.     

Controlled Ring‐Opening Polymerization of O‐Carboxyanhydrides Using a β‐Diiminate Zinc Catalyst

Recently O‐carboxyanhydrides (OCAs) have emerged as a class of viable monomers which can undergo ring‐opening polymerization (ROP) to prepare poly(α‐hydroxyalkanoic acid) with functional groups that are typically difficult to achieve by ROP of lactones. Organocatalysts for the ROP of OCAs, such as dimethylaminopyridine (DMAP), may induce undesired epimerization of the α‐carbon atom in polyesters resulting in the loss of isotacticity. Herein, we report the use of (BDI‐IE)Zn(OCH(CH₃)COOCH₃) ((BDI)Zn‐1, (BDI‐IE)=2‐((2,6‐diethylphenyl)amino)‐4‐((2,6‐diisopropylphenyl)imino)‐2‐pentene), for the controlled ROP of various OCAs without epimerization. Both homopolymers and block copolymers with controlled molecular weights, narrow molecular weight distributions, and isotactic backbones can be readily synthesized. (BDI)Zn‐1 also enables controlled copolymerization of OCAs and lactide, facilitating the synthesis of block copolymers potentially useful for various biomedical applications. Preliminary mechanistic studies suggest that the monomer/dimer equilibrium of the zinc catalyst influences the ROP of OCAs, with the monomeric (BDI)Zn‐1 possessing superior catalytic activity for the initiation of ROP in comparison to the dimeric (BDI)Zn complex.     

Structure of wyosine, the condensed tricyclic nucleoside of torula yeast phenylalanine transfer ribonucleic acid

Large-scale isolation of the minor nucleoside wyosine of torula yeast tRNA(Phe) was accomplished by a combination of enzymatic digestion and reversed-phase chromatography: the wyosine-containing nucleotide fraction, which was obtained by partial digestion of unfractionated tRNA (1 g) with nuclease P1, was concentrated by reversed-phase column chromatography followed by complete digestion with nuclease P1/alkaline phosphatase. The nucleoside mixture thus obtained was purified by reversed-phase HPLC, providing wyosine (70 microg). Comparison of this nucleoside with a chemically synthesized authentic sample has unambiguously established that the structure of wyosine is 4,6-dimethyl-3-beta-D-ribofuranosyl-3,4-dihydro-9H-imidazo[1,2-a]purin-9-one (2).     

基于Fe_3O_4/乙二胺四乙酸磁性粒子的集成化蛋白质组学方法

建立了基于Fe_3O_4/乙二胺四乙酸(EDTA)磁性粒子的集成化蛋白质组学研究方法。首先用共沉淀法合成EDTA负载的Fe_3O_4/EDTA磁性粒子。在优化的溶液条件下(95%乙腈-1%三氟乙酸,体积分数),100μg Fe_3O_4/EDTA磁性粒子可吸附12.4μg牛血清白蛋白(BSA),吸附容量是商品化磁珠的10倍左右。以BSA作为标准蛋白质,对所合成的Fe_3O_4/EDTA磁性粒子作为蛋白质组学反应器的酶解时间进行了优化,发现Fe_3O_4/EDTA磁性粒子处理BSA酶解1、8和16 h的肽段序列覆盖率和特征肽段结果相当。因此,可以将复杂的蛋白质样品前处理时间缩短至2 h内。最后,将所合成的Fe_3O_4/EDTA磁性粒子应用于血清的蛋白质组学研究,成功地鉴定出218种蛋白质,其中包含了41种美国食品药品管理局(FDA)认证的生物标志物。所发展的基于Fe_3O_4/EDTA磁性粒子的蛋白质组学样品前处理方法将蛋白质样品预富集、还原、烷基化、酶解、多肽除盐和洗脱等步骤集成到一起,减少了样品转移和处理所造成的损失。这种技术具有快速、灵敏和易于操作的特点,可用于临床蛋白质组学研究。     

Determination of phytic acid and inositolphosphates in barley

Phytic acid (PA) and lower inositolphosphates (InsP(n) ) is the main storage form of phosphorus in grains or seeds. The content of PA and InsP(n) in different varieties of barley was analyzed by capillary isotachophoresis and online-coupled capillary isotachophoresis with CZE. The electrolytes (in demineralized water) for the isotachophoretic analysis consisted of 10?mM HCl, 14?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading) and 10?mM citric acid (terminating). The optimized electrolytes for the online coupling isotachophoresis with zone electrophoresis analysis were mixtures of 5?mM HCl, 7?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading), 20?mM citric acid, 10?mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (background) and 10?mM citric acid (terminating). PA and all studied InsP(n) were separated within 25?min and detected by a conductivity detector. Simple sample preparation (acidic extraction), sufficient sensitivity, speed of analysis, and low running cost are important attributes of the electrophoretic methods. The method was used for the determination of PA and InsP(n) in barley varieties within an ongoing research project.     

Enantioseparation of dansyl amino acids by ligand‐exchange capillary electrophoresis with zinc(II)‐L‐phenylalaninamide complex

A novel method of chiral ligand‐exchange CE was developed with L ‐amino acylamides as a chiral ligand and zinc(II) as a central ion. It has been demonstrated that these chiral complexes, such as Zn(II)‐L ‐alaninamide, Zn(II)‐L ‐prolinamide, and Zn(II)‐L ‐phenylalaninamide, are suitable for use as chiral selectors for the enantioseparation of either individual pair of or mixed dansyl amino acids. The optimal separation running buffer consisted of 5 mM ammonium acetate, 100 mM boric acid, 4 mM ZnSO₄·7 H₂O, and 8 mM L ‐amino acylamides at pH 8.2. The experiments showed that apart from the effect of the concentration of the complexes on the resolution and the migration time, the buffer pH also had a sharp influence on resolution. The employed chiral ligands exhibited different enantioselectivities and enantiomer migration orders. D ‐Amino acids migrate faster than L ‐amino acids when Zn(II)‐L ‐alaninamide and Zn(II)‐L ‐phenylalaninamide are used as chiral selectors, but it was observed that the migration order is reversed when Zn(II)‐L ‐prolinamide is used as the chiral selector. Furthermore, the amount of dansylated amino acids is found to be highly dependent on the labeling temperature.     

Design of a colicin E7 based chimeric zinc-finger nuclease

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.     

Several modified piezoelectric quartz crystals for determination of omethoate

A chromatographic method for the determination of transition metals in human hair samples is described. The method involves the separation of Cu, Pb, Zn, Ni, Co, Cd and Mn in a C18 column coated with sodium hexadecane-sulfonate (SHS) and spectrophotometric detection (520 nm) after post-column reaction of the eluted metals with 4-(2-pyridylazo)-resorcinol (PAR). The eluent was a 100 mM tartrate solution adjusted to pH 3.1 with a 2 M sodium hydroxide solution (flow-rate=1.0 ml min(-1)). A good separation of the eluted metals (specially for Cu/Pb and Zn/Ni) has been achieved. The detection limits, expressed as mug l(-1), were 2.2 (Cu), 8.0 (Pb), 2.8 (Zn), 1.5 (Ni), 1.5 (Co), 12.0 (Cd), and 1.4 (Mn). A microwave-assisted closed vessel acid digestion procedure with HNO(3)+HClO(4) (4+1 ml) was used for the hair samples solubilisation. Nineteen hair samples were analysed with the proposed method. The results were in good agreement with those obtained by atomic absorption spectrometry (AAS).