Synthesis and characterization of N-octanoyl-beta-D-glucosylamine, a new surfactant for membrane studies

The new nonionic glycosidic surfactant N-octanoyl-beta-D-glucosylamine (NOGA, molar mass 305.37 g) was synthesized through an easy and efficient two-step procedure. Specifically, beta-D-glucosylamine was obtained by the replacement of the anomeric hydroxyl of D-glucose by an amino group which was then selectively acylated. NOGA was finally purified by silica gel column chromatography and recrystallization. This compound is stable and soluble in water and usual buffers up to 80 mM at 4 degrees C and up to 0.2 M at 37 degrees C. NOGA solutions are also characterized by a low ultraviolet light absorbance above 250 nm (epsilon 280 approximately 1.5 M-1 cm-1). Due to its very high critical micelle concentration (CMC = 80 mM, as determined by spectrofluorimetry), this surfactant may easily be removed from samples by dialysis or, to a lesser extent, by adsorption onto hydrophobic beads. Furthermore, NOGA is colorimetrically titrable by the ninhydrin method and its weak interference in protein determination by the bicinchoninic acid method is easy to overcome. This surfactant exhibits a good solubilizing power toward membrane proteins, with a marked selectivity for spiralin, a bacterial surface antigen. Protein extraction started below the CMC, but was much more effective above this concentration threshold. NADH oxidase activity, ligand binding by the glycine betaine-binding protein, and antigenicity of more than 20 membrane or soluble proteins were not altered by NOGA. Thus, owing to its extraction efficacy and mildness toward protein structure and activity, NOGA should prove useful for membrane studies and offers the additional advantage of being easy to synthesize at low cost.     

Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA

The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.     

新型含银配合物的合成与表征

合成了一种由银直接连接Anderson型钼氧酸盐阴离子形成的新型二维配合物,并用单晶X射线衍射仪测定了其晶体结构,用元素分析仪及红外光谱等进一步表征了化合物的结构。含银配合物为三斜晶系,P-1空间群,晶胞参数为a=1.1676(3)nm,b=1.2205(4)nm,c=1.2566(6)nm;α=95.313(5)°,β=113.974(5)°,γ=117.079(3)°;V=1.3695(9)nm3,Z=1。配合物中,相邻的4个{Fe(OH)6Mo6O18}3-之间存在两种连接方式,一种是被孤立的{AgO6}八面体通过端氧沿b轴连接起来;另一种则是被2个共边相连的{AgO6}八面体通过端氧沿b轴桥连起来。这两种连接方式沿c轴方向交替排列形成了二维网状结构。     

Cloning and characterization of the gene encoding a new DNA methyltransferase from Neisseria gonorrhoeae

A HindIII fragment of N. gonorrhoeae MS11 DNA coding for DNA methyltransferase (MTase) activity was cloned and expressed in E. coli AP1-200-9 cells. The sequence of 4681 bp was determined, and its analysis revealed two open reading frames (ORFs) sharing some similarity with known DNA MTases. ORF1 encodes an active N4mC MTase (M.NgoMV). The enzyme modifies only one strand of double stranded DNA and preferentially recognises the sequence GCCHR although it is able to methylate other sites. The exact recognition sequence cannot be precisely defined due to a relaxed specificity. The second ORF shows high homology to 5mC Mtases, but we were unable to demonstrate DNA methylating activity of its product either in vivo or in vitro.     

Unveiling two distinct ribonuclease activities and a topoisomerase activity in a site-specific DNA recombinase

The site-specific DNA recombinase Flp shows two types of RNA cleavage activities on hybrid DNA-RNA substrates. One targets the phosphodiester position involved in DNA recombination and follows a related mechanistic path. In this two-step reaction, first-strand scission is mediated by a nucleophilic attack of the scissile phosphodiester bond by the active site tyrosine of Flp. The resultant 3'-O-phosphoryl tyrosine bond is then attacked by the adjacent 2'-hydroxyl group. The second activity targets the immediately adjacent phosphodiester bond to the 3' side using a distinct mechanism. In this reaction, the vicinal 2'-hydroxyl directly attacks the phosphate group in a manner that is reminiscent of the pancreatic RNase mechanism. The Flp protein can also be shown to possess a topoisomerase-like activity.     

2-羟基-5-特辛基苯乙酮肟的合成及表征

以四氯乙烯为溶剂,对特辛基苯酚、乙酰氯、无水AlCl3和盐酸羟胺为主要原料,采用分步合成法合成了2-羟基-5-特辛基苯乙酮肟.考察了无水AlCl3用量、乙酰氯的用量、Fries重排温度、肟化反应盐酸羟胺用量以及缚酸剂无水碳酸钠用量对产物产率的影响,实验结果表明,当n(AlCl3)∶n(乙酰氯)∶n(对特辛基苯酚)=1.5∶2∶1,Fries重排温度为120℃,n(Na2CO3)∶n(盐酸羟胺)∶n(对特辛基苯酚)=0.5∶1∶1.产物收率64.9%.对合成产物利用核磁共振进行了结构表征.     

Efficient modification of the APRT gene by FLP/FRT site-specific targeting

The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5 X 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.     

Synthesis and analgesic properties of new 5-thioaryl pyrazole derivatives

A new series 5-thio aryl pyrazole derivatives were proposed aiming analgesic activity. In this work, 8 new compounds of this class were synthesized using usual synthetic methodology, having as key intermediate the 3-methyl-4-nitro-5-chloropyrazole-1-phenyl derivative and subsequent reaction with several nucleophiles sulfides. Pharmacological evaluation of this series showed analgesic activity in the some extent in especially for 5-(4-bromophenyl)-thio-3-methyl-4-nitro-1-phenylpyrazole which was the most potent in this series, presenting an analgesic action comparable to that show by dipyrone.     

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.     

Exploring a channel to the active site of copper/topaquinone-containing phenylethylamine oxidase by chemical modification and site-specific mutagenesis

Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.     

Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.     

Synthesis and characterization of new zirconia-based polymeric cation-exchange stationary phases for high-performance liquid chromatography of proteins

Ion-exchange chromatography is a major method used for large-scale protein separations. New zirconia-based polymeric cation-exchange HPLC stationary phases have been developed for protein separations. Two routes were employed for the synthesis. In one method, polyethyleneimine (PEI) was adsorbed onto porous zirconia particles and cross-linked with 1,4-butanediol diglycidyl ether (BUDGE). Succinic anhydride was then reacted with the remaining primary and secondary amine groups on PEI to afford anionic functionalities. The second method utilizes poly(acrylic acid) anhydride as both the crosslinker and the stationary phase. The resulting stationary phases act to separate proteins by a weak cation-exchange mechanism with a slight contribution to retention from hydrophobic interactions. In the presence of 20 mM phosphate buffer, Lewis acid/base interactions between the zirconia support and the proteins, which can significantly broaden the peaks, are sufficiently suppressed. The effects of ionic strength, mobile phase pH, and salt type are discussed. Protein mass recovery and loading capacity for protein separations on these phases have been evaluated. These weak cation-exchange stationary phases exhibit good stability under normal separation conditions for months and are stable in alkaline solution up to pH 10. In contrast to zirconia supports modified with small anionic species, these new phases have no limitation on the type of salt used as the eluent, and they exhibit unique selectivities. Therefore, they offer interesting alternatives for protein separations. To our knowledge, this work represents the first successful example of protein separations using porous zirconia-based polymeric phases under normal chromatographic conditions, which will definitely help make zirconia-based supports more useful for bio-separation.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.     

Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis

Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive. In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity. A modification of the quantitative DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, OR-flanking lambda DNA by cI repressor oligomers. With the exception of one mutant, only those repressors capable of octamerizing were found to exhibit non-specific DNA-binding activity. The non-specific interaction was accurately modeled using either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach. It was found that non-specific DNA-binding by repressor oligomers is highly cooperative and energetically independent from site-specific binding at OR. Furthermore, the coupling free energy resolved for non-specific binding was similar to that of site-specific binding for each repressor, suggesting that similar structural elements may mediate the cooperative component of both binding processes. It is proposed that the state of assembly of the repressor molecule modulates its relative affinity for specific and non-specific DNA sequences. These specificities are allosterically regulated by the transmission of assembly-state information from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding. While dimers have a high affinity for their cognate sites within OR, tetramers and octamers may preferentially recognize non-specific DNA sequences. The concepts and findings developed in this study may facilitate quantitative characterization of the relationships between specific, and non-specific binding in other systems that utilize multiple modes of DNA-binding cooperativity.     

超分散剂的合成及其在改性无机纳米粉体中的应用

针对无机纳米粉体的表面改性,以自由基聚合的方法制备以马来酸酐及其单酯物为锚固基团、甲基丙烯酸丁酯(BMA)为溶剂化链、苯乙烯(St)为功能基团的超分散剂SMB.研究不同超分散剂种类、用量以及传统分散剂改性无机纳米粉末的效果,改性前后纳米粉末通过亲油化度、润湿性检测以及SEM和TEM观察以表征其改性效果.研究表明,超分散剂的适宜用量为8％;超分散剂SMB-2改性纳米TiO2粉体的改性效果较好;通过对比超分散剂与传统改性剂钛酸酯、硅烷偶联剂和TDI改性纳米TiO2粉体的改性效果可知,超分散剂的改性效果较佳.     

An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.