Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Chemistry ; 27(39): 10058-10067, 2021 Jul 12.
Article in English | MEDLINE | ID: mdl-33991022

ABSTRACT

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.


Subject(s)
Aptamers, Nucleotide , Electrophoresis, Capillary , High-Throughput Nucleotide Sequencing , Reproducibility of Results , Thrombin
2.
Electrophoresis ; 41(13-14): 1152-1159, 2020 07.
Article in English | MEDLINE | ID: mdl-32253765

ABSTRACT

A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.


Subject(s)
Electrophoresis, Capillary/methods , Fluorescent Dyes/chemistry , Fluorescent Dyes/isolation & purification , Isotachophoresis/methods , Anions , Aptamers, Nucleotide
3.
Chem Commun (Camb) ; 54(56): 7822-7825, 2018 Jul 10.
Article in English | MEDLINE | ID: mdl-29947371

ABSTRACT

The induced-fit accommodation of a variety of gaseous molecules including non-polar molecules has been demonstrated in porphyrin-based supramolecular architectures for the first time. Moreover, the gas uptake behaviour can be modulated by changing the central cation of porphyrin.

4.
Nat Commun ; 8(1): 109, 2017 07 24.
Article in English | MEDLINE | ID: mdl-28740078

ABSTRACT

Despite recent advances in the carbonization of organic crystalline solids like metal-organic frameworks or supramolecular frameworks, it has been challenging to convert crystalline organic solids into ordered carbonaceous frameworks. Herein, we report a route to attaining such ordered frameworks via the carbonization of an organic crystal of a Ni-containing cyclic porphyrin dimer (Ni2-CPDPy). This dimer comprises two Ni-porphyrins linked by two butadiyne (diacetylene) moieties through phenyl groups. The Ni2-CPDPy crystal is thermally converted into a crystalline covalent-organic framework at 581 K and is further converted into ordered carbonaceous frameworks equipped with electrical conductivity by subsequent carbonization at 873-1073 K. In addition, the porphyrin's Ni-N4 unit is also well retained and embedded in the final framework. The resulting ordered carbonaceous frameworks exhibit an intermediate structure, between organic-based frameworks and carbon materials, with advantageous electrocatalysis. This principle enables the chemical molecular-level structural design of three-dimensional carbonaceous frameworks.Carbon-based materials are promising alternatives to noble metal catalysts, but their structures are typically disordered and difficult to control. Here, the authors obtain ordered carbonaceous frameworks with advantageous electrocatalytic properties via the carbonization of nickel-containing porphyrin dimer networks.

5.
Article in English | MEDLINE | ID: mdl-27419901

ABSTRACT

The sensitivity, range of applications, and reaction mechanism of 2-hydrazinoquinoline as a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were examined. Using a reaction chamber (125L) equipped with a stirring fan and a window for moving the MALDI-MS plate and volatile samples in and out, the sensitivities of 2-hydrazinoquinoline to gaseous aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and n-butyraldehyde) and ketones (acetone, methyl ethyl ketone, and methyl isobutyl ketone) were determined to be at least parts per million (ppm) levels. On the other hand, carboxylic acids (formic acid, acetic acid, propionic acid, and butyric acid) and esters (ethyl acetate, pentyl acetate, isoamyl acetate, and methyl salicylate) could not be detected by 2-hydrazinoquinoline in MALDI-MS. In addition to 2,4-dinitrophenylhydrazine, a common derivatization reagent for analyzing carbonyl compounds quantitatively in gas chromatography and liquid chromatography, the dissolution of 2-hydrazinoquinoline in an acidic solution, such as trifluoroacetic acid, was essential for its function as a reactive matrix for MALDI- MS.

6.
Comput Intell Neurosci ; 2016: 7267691, 2016.
Article in English | MEDLINE | ID: mdl-27217825

ABSTRACT

We observed spike trains produced by one-shot electrical stimulation with 8 × 8 multielectrodes in cultured neuronal networks. Each electrode accepted spikes from several neurons. We extracted the short codes from spike trains and obtained a code spectrum with a nominal time accuracy of 1%. We then constructed code flow maps as movies of the electrode array to observe the code flow of "1101" and "1011," which are typical pseudorandom sequence such as that we often encountered in a literature and our experiments. They seemed to flow from one electrode to the neighboring one and maintained their shape to some extent. To quantify the flow, we calculated the "maximum cross-correlations" among neighboring electrodes, to find the direction of maximum flow of the codes with lengths less than 8. Normalized maximum cross-correlations were almost constant irrespective of code. Furthermore, if the spike trains were shuffled in interval orders or in electrodes, they became significantly small. Thus, the analysis suggested that local codes of approximately constant shape propagated and conveyed information across the network. Hence, the codes can serve as visible and trackable marks of propagating spike waves as well as evaluating information flow in the neuronal network.


Subject(s)
Action Potentials/physiology , Models, Neurological , Nerve Net/physiology , Neurons/physiology , Animals , Cell Culture Techniques , Electric Stimulation , Embryo, Mammalian , Hippocampus/cytology , Rats , Rats, Wistar
7.
Chem Commun (Camb) ; 51(9): 1685-8, 2015 Jan 31.
Article in English | MEDLINE | ID: mdl-25512950

ABSTRACT

Linear assemblies of a 1 : 1 porphyrin-fullerene C60 complex were formed in vertical cylindrical polyether nanodomains of amphiphilic block copolymer films by a simple spin coating-annealing method. The nanocylinder structures were retained even with high contents of the complex in the polymer films.

8.
J Org Chem ; 79(7): 2980-92, 2014 Apr 04.
Article in English | MEDLINE | ID: mdl-24597606

ABSTRACT

Free-bases and a nickel(II) complex of phenothiazine-bridged cyclic porphyrin dimers bearing self-assembling 4-pyridyl groups (M2-Ptz-CPDPy(OCn); M = H2 or Ni, OCn = OC6 or OC3) at opposite meso-positions have been prepared as host molecules for fullerenes. The free-base dimer (H4-Ptz-CPDPy(OC6)) includes fullerenes with remarkably high association constants such as 3.9 ± 0.7 × 10(6) M(-1) for C60 and 7.4 ± 0.8 × 10(7) M(-1) for C70 in toluene. This C60 affinity is the highest value ever among reported receptors composed of free-base porphyrins. The nickel dimer (Ni2-Ptz-CPDPy(OC6)) also shows high affinities for C60 (1.3 ± 0.2 × 10(6) M(-1)) and C70 (over 10(7) M(-1)). In the crystal structure of the inclusion complex of C60 within H4-Ptz-CPDpy(OC3), the C60 molecule is located just above the centers of the porphyrins. The two porphyrin planes are almost parallel to each other and the center-to-center distance (12.454 Å) is close to the optimal separation (∼12.5 Å) for C60 inclusion. The cyclic porphyrin dimer forms a nanotube through its self-assembly induced by C-H···N hydrogen bonds between porphyrin ß-CH groups and pyridyl nitrogens as well as π-π interactions of the pyridyl groups. The C60 molecules are linearly arranged in the inner channel of this nanotube.

9.
APMIS ; 118(3): 167-78, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20132182

ABSTRACT

Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two-component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two-dimensional gel electrophoresis (2-DE) and pulsed-field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two-component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2-DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection.


Subject(s)
Antigens, Bacterial/analysis , Bacterial Outer Membrane Proteins/analysis , Carrier Proteins/analysis , Pharyngitis/microbiology , Streptococcus pyogenes/chemistry , Electrophoresis, Gel, Pulsed-Field , Electrophoresis, Gel, Two-Dimensional , Histidine Kinase , Humans , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Mutation , Streptococcus pyogenes/genetics
10.
Antimicrob Agents Chemother ; 54(1): 98-102, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19805566

ABSTRACT

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic shock syndrome. However, the prevalence of CLI-resistant Streptococcus pyogenes strains is increasing worldwide, and the effect of CLI on CLI-resistant S. pyogenes strains remains unknown. We aimed to evaluate the effect of CLI on the in vitro production of three major virulent exoproteins, namely, streptolysin O (Slo), NAD glycohydrolase (Nga), and streptokinase (Ska), by CLI-resistant S. pyogenes strains. After the incubation of M1 serotype CLI-resistant S. pyogenes D2TY in the presence of 1 microg/ml CLI, the amounts of Slo, Nga, and Ska and the levels of slo, nga, and ska mRNA in the supernatant were analyzed by Northern blotting and Western blotting, respectively. The results of both assays showed that the production of Slo, Nga, and Ska was higher with CLI treatment than without CLI treatment. We evaluated the role of the sensor kinase CovS, which is involved in the two-component system of S. pyogenes, in the CLI-induced production of these three exoproteins. Northern blotting analysis revealed that CLI induced the expression of covS mRNA in wild-type strain D2TY. Furthermore, both Northern blotting and Western blotting analyses showed that CLI decreased the levels of expression of Slo, Nga, and Ska in isogenic covS mutant D2TYcovS. These results suggest that CLI increases the production of three virulent exoproteins in CLI-resistant S. pyogenes strains via the action of CovS.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clindamycin/pharmacology , Genes, Bacterial/genetics , Intracellular Signaling Peptides and Proteins/genetics , NAD+ Nucleosidase/biosynthesis , NAD+ Nucleosidase/genetics , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/genetics , Streptokinase/biosynthesis , Streptokinase/genetics , Streptolysins/biosynthesis , Streptolysins/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Blotting, Northern , Blotting, Western , Drug Resistance, Bacterial/genetics , Gene Expression Regulation, Bacterial/drug effects , Histidine Kinase , Lincosamides/pharmacology , Microbial Sensitivity Tests , Mutation , RNA, Bacterial/biosynthesis , RNA, Bacterial/genetics , Reverse Transcriptase Polymerase Chain Reaction , Streptococcus pyogenes/enzymology
11.
Antimicrob Agents Chemother ; 51(2): 461-7, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17101685

ABSTRACT

The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD+ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS- or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Clindamycin/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Streptococcus pyogenes/drug effects , Humans , NAD+ Nucleosidase/biosynthesis , Streptococcal Infections/drug therapy , Streptococcal Infections/microbiology , Streptococcus pyogenes/growth & development , Streptococcus pyogenes/metabolism , Streptolysins/biosynthesis
SELECTION OF CITATIONS
SEARCH DETAIL
...