Synthesis and Properties of Copolymers Having Polyconjugated Blocks in the Macromolecules. IV. Use of Aromatic N,N′-Bis(nitrosoacetyl)diamines for the Synthesis of Carborane-Containing Polymers

N-Nitrosoacetylam inophenyl-o-carboranes have been used for the synthesis of polyconjugated polyazophenylene and styrene copolymers containing carborane units. In the presence of carborane-containing polymers, destruction of thermostable polymers in air at high temperatures becomes thermal destruction due to processes of nonchain inhibition.     

Photoinduced orientational ordering in the series of methacrylic azopolymers

Polymethacrylates with side azo fragments containing various end substituents and spacers are synthesized and characterized. Spatial ordering of azo fragments in the series of the synthesized azo polymers under the action of polarized excitation light is studied by the methods of null ellipsometry and polarization spectroscopy. Two types of anisotropy development during photoirradiation are found. In homologs with strong acceptor substituents of azo fragments, biaxial orientational structure can be observed; as the radiation dose is increased, this structure is transformed into a uniaxial structure with a negative order parameter and with an axis parallel to the polarization vector of the excitation light. In this structure, the maximum degree of orientational order is achieved in polymers with high concentrations of azo fragments. In homologs with donor substituents of azo fragments, the initial stages of irradiation likewise lead to the development of biaxial orientation; later, this orientation is transformed into an isotropic distribution. The mode of anisotropy development is controlled by the lifetime of photoisomers of azo fragments.     

Residue preference mapping of ligand fragments in the Protein Data Bank

The interaction between small molecules and proteins is one of the major concerns for structure-based drug design because the principles of protein-ligand interactions and molecular recognition are not thoroughly understood. Fortunately, the analysis of protein-ligand complexes in the Protein Data Bank (PDB) enables unprecedented possibilities for new insights. Herein, we applied molecule-fragmentation algorithms to split the ligands extracted from PDB crystal structures into small fragments. Subsequently, we have developed a ligand fragment and residue preference mapping (LigFrag-RPM) algorithm to map the profiles of the interactions between these fragments and the 20 proteinogenic amino acid residues. A total of 4032 fragments were generated from 71?798 PDB ligands by a ring cleavage (RC) algorithm. Among these ligand fragments, 315 unique fragments were characterized with the corresponding fragment-residue interaction profiles by counting residues close to these fragments. The interaction profiles revealed that these fragments have specific preferences for certain types of residues. The applications of these interaction profiles were also explored and evaluated in case studies, showing great potential for the study of protein-ligand interactions and drug design. Our studies demonstrated that the fragment-residue interaction profiles generated from the PDB ligand fragments can be used to detect whether these fragments are in their favorable or unfavorable environments. The algorithm for a ligand fragment and residue preference mapping (LigFrag-RPM) developed here also has the potential to guide lead chemistry modifications as well as binding residues predictions.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Characterization of the selective staining of DNA on gels using two fluorochromes

Ethidium bromide (EB) and 4'-6-diamidino-2-phenylindole (DAPI) are both well-known fluorochromes for detecting DNA fragments. EB binds to DNA by intercalation and DAPI binds in the DNA minor groove. We previously developed a staining method using both EB and DAPI that is selective for AT-rich DNA fragments. Using this double-staining method, AT-rich DNA fragments are visualized as bluish-white fluorescent bands. To further characterize this method, a series of synthetic DNA fragments were designed with systematic variations in the length, AT content, and DNA sequence pattern. The staining properties of these fragments were determined in the presence of DAPI and EB, and the following results were obtained. (i) In a series of fragments with three AT base pairs followed by one GC base pair, the stained DNA fragments exhibited different fluorescent colors and varied from bluish (more DAPI staining) to pinkish (less DAPI staining) in the order 5'-AAA-3', 5'-AAT-3', 5'-ATA-3', 5'-TTA-3'. (ii) In fragments with constant AT content, the blue fluorescent color increased with increasing number of A (or T) nucleotides, due to increased DAPI binding. The blue color was saturated when the number of A (or T) nucleotides was 12 or greater. (iii) The fluorescent color of the stained DNA fragments changed in the order of red-orange, pink, pinkish-white, white, bluish-white, blue as the AT content increased from 0 to 100%. Thus, the fluorescent color of DNA fragments stained with DAPI and EB depends on base composition and nucleotide sequence, suggesting that individual stained DNA fragments may have characteristic and specific fluorescent colors. The fluorescent color emitted by specific stained DNA fragments in the presence of EB and DAPI can be analyzed with a high degree of sensitivity and resolution using the XYZ colorimetric system.     

The role of water molecules in the interfragmental charge transfer of the protolytic forms of fluoroquinolones in the ground and excited states

Quantum-chemical analysis (TD-DFT/B3LYP/6-31G) of the influence of H-bonding in the donor-acceptor fluoroquinolone compounds with the water molecules on the interfragmentary charge transfer was carried out. The values of charge transfer between the molecular fragments in the transition from the ground into the singlet excited state were calculated. It was found that, depending on the protolytic form of a compound, the frontier molecular orbitals are localized on different fragments, which leads to the specificity of the formation of the electron density transfer channel. The influence of individual substituents in the quinolones on the localization of particular MOs is relatively insignificant.     

Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography

Ion pair reverse phase high performance liquid chromatography on non-porous alkylated poly(styrene-divinylbenzene) particles enables the high resolution separation of double stranded DNA fragments. To further understand the separation mechanisms involved in ion pair reverse phase liquid chromatography we have analysed the effects of curved or "bent" DNA fragments with respect to their separation using both gel electrophoresis and ion pair reverse phase liquid chromatography. Size dependent separations of curved DNA fragments that migrate anomalously during gel electrophoresis were observed using ion pair reverse phase liquid chromatography. To further study the sequence effect and resulting changes in hydrophobicity of the duplex DNA, PCR fragments were generated that contain uracil in place of thymine. The resulting fragments were shown to elute with shorter retention times, demonstrating that sequence-specific effects can alter the retention of duplex DNA. The study was extended to the investigation of non-canonical B-DNA structures (Holliday junctions) under various chromatographic conditions, demonstrating that the coaxial stacking of the helices in such structures, in the presence of magnesium causes a change in retention.     

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.     

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered extracellular striatal in vivo biotransformation of the opioid neuropeptide dynorphin A(1-17) in the unilateral 6-OHDA rat model of Parkinson's disease

The in vivo biotransformation of dynorphin A(1-17) (Dyn A) was studied in the striatum of hemiparkinsonian rats by using microdialysis in combination with nanoflow reversed-phase liquid chromatography/electrospray time-of-flight mass spectrometry. The microdialysis probes were implanted into both hemispheres of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats. Dyn A (10 pmol microl(-1)) was infused through the probes at 0.4 microl min(-1) for 2 h. Samples were collected every 30 min and analyzed by mass spectrometry. The results showed for the first time that there was a difference in the Dyn A biotransformation when comparing the two corresponding sides of the brain. Dyn A metabolites 1-8, 1-16, 5-17, 10-17, 7-10 and 8-10 were detected in the dopamine-depleted striatum but not in the untreated striatum. Dyn A biotransformed fragments found in both hemispheres were N-terminal fragments 1-4, 1-5, 1-6, 1-11, 1-12 and 1-13, C-terminal fragments 2-17, 3-17, 4-17, 7-17 and 8-17 and internal fragments 2-5, 2-10, 2-11, 2-12, and 8-15. The relative levels of these fragments were lower in the dopamine-depleted striatum. The results imply that the extracellular in vivo processing of the dynorphin system is being disturbed in the 6-OHDA-lesion animal model of Parkinson's disease.     

磷酸化对D1蛋白7900和9300 降解片段的影响

利用Western blotting技术对未磷酸化和磷酸化光合系统Ⅱ(PSⅡ)膜中D1蛋白的7900和9300降解片段进行了检测. 结果表明, 超氧阴离子、 过氧化氢和羟基自由基参与了未磷酸化和磷酸化D1蛋白的7900和9300片段的产生. 磷酸化能够部分抑制D1蛋白7900和9300片段的产生, 从而对D1蛋白起到保护作用.     

Mannitol influence on the separation of DNA fragments by capillary electrophoresis in entangled polymer solutions

A new kind of sieving matrix is presented in this paper to allow satisfactory separation of DNA fragments in a relatively low viscous solution. When a certain amount of mannitol was added to cellulose solution not concentrated enough to separate PGEM-3Zf(+)/HaeIII standards well, a polymer solution with low viscosity but with very good separation effects was obtained. The separation result of this sieving buffer was comparable with those using highly concentrated cellulose solutions. The sieving ability of solutions with different cellulose concentrations and different amounts of mannitol has been investigated. It was proved that 0.5% was the minimum hydroxypropylmethylcellulose (HPMC) concentration that could be used to separate DNA fragments satisfactorily. HPMC solutions with a concentration of less than 0.5% could not separate the standard DNA fragments even in the presence of mannitol. It was found that 6% was the optimized mannitol concentration because either more or less mannitol will lead a decrease of resolution. The principle of the positive influence of mannitol has also been discussed.     

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments**

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.     

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.