DNA sensor: A novel electrochemical gene detection method using carbon electrode immobilized DNA probes

Abstract

The authors have developed a novel, rapid, convenient, and specific gene detection method, named the ‘DNA sensor,’ using a graphite electrode loaded with DNA probes. Synthesized oligonucleotide (5-TGCAGTTCCGGTGGCTGATC-3′) complementary to oncogene v-myc was employed for a model probe. The oligonucleotide was chemically adsorbed on a basal plane pyrolytic graphite (BPPG) electrode. The sensor was able to be applied to a hybridization reaction (40°C) in a linearized pVM623 solution carrying the Pst I fragment of v-myc (1.5 kbp).

After the hybridization reaction, the sensor was immersed into an acridine orange solution (1 μM) and washed with a phosphate buffer (pH 7.0). Acridine orange intercalated between base pairs of the formed double stranded DNAs on the electrode. The anodic peak potential of acridine orange that interacted with the DNAs on the electrode was measured. The positive shift of the peak potential increased in proportional to the pVM623 concentration in the hybridization reaction. 10^?1 g/ml of pVM623 was able to be detected in the buffer solution using the sensor. This gene detection was completed within an hour.     

Discovery of Oxysterol-Derived Pharmacological Chaperones for NPC1: Implication for the Existence of Second Sterol-Binding Site

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Highlights? Niemann-Pick disease type C is caused by folding defect in NPC1 protein ? Oxysterols and chemically optimized derivatives act as pharmacological chaperones ? Defects in localization, stability, maturation, and function were corrected in cell ? These derivatives bind to second sterol-binding site on NPC1 protein     

Fully automated reagent peak‐free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column‐switching system and fluorous scavenging derivatization

In this study, we combined a column‐switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak‐free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1‐pyrenemethylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and 1‐hydroxy‐1H‐benzotriazole. Residual excess of the unreacted reagent was tagged with 2‐(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column‐switching system placed in front of an analytical reversed‐phase column. The signal of the fluorous‐tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type‐II model mice and their control.     

Synthesis of novel 4′-C-methyl-1′,3′-dioxolane pyrimidine nucleosides and evaluation of its anti-HIV-1 activity

The key glycosyl donor for the target molecule 12 was prepared by two-step sequences; (1) acetalization of tert-butyldimethylsilyloxyacetaldehyde with 3-bromopropanediol, (2) DBN-initiated β-elimination of the resulting 2-(tert-butyldimethylsilyloxy)methyl-4-bromomethyl-1,3-dioxolane 11. Electrophilic glycosidation between 12 and silylated pyrimidine nucleobase proceeded efficiently to provide a mixture of β- and α-anomers of the respective glycosides 14 and 15. Tin radical-mediated reduction of the bromomethyl functional group of 14 and 15 gave protected 4′-C-methyl-dioxorane uracil- 16 and thymine nucleoside 17. The respective cytosine nucleoside 18 was synthesized from 16. De-silylation of 4′-methyl-1′,3′-dioxolane pyrimidine nucleosides 16–18 gave the target molecules. Evaluation of the anti-HIV-1 activity of the β- and α-anomers of the novel 4′-C-methyl-1′,3′-dioxolane nucleosides 22β,α–24β,α revealed that none of the nucleoside derivatives possess anti-viral activity against HIV-1 and show cytotoxicity against MT-4 cells at 100 μM.     

Invited Lecture. Ferroelectric liquid crystalline polymers and related model compounds with a low–moderate degree of polymerization

Abstract

Ferroelectric liquid crystalline polymers (FLCPs) with a low-moderate degree of polymerization were synthesized. These had a comb structure; the main chain was polyacrylate and the side chain consisted of a flexible spacer, a core and an optically active chiral end group. They exhibited electro-optic switching times ranging from a few milliseconds to a few seconds in the S*_c phase. As the molecular weight M _n increased, the range of the S*_c shifted to higher temperatures. At a given temperature, the switching time increased with M _n .

Spontaneous polarizations P _s , apparent cone angles 2θ, electro-optic switching times τ and rotational viscosities η of some polyoxyethylene FLCPs and corresponding low molecular weight ferroelectric liquid crystal materials (FLCs) were also measured. This is the first report of polyoxyethylene FLCPs. There was no significant difference in P _s and 2θ, which shows that the arrangement of the side chains in the FLCP is similar to that of the molecules in ordinary FLC. On the contrary, τ and η for the FLCPs were 10²–10³ times as large as those for FLCs. The rotational viscosity of the FLCP was mainly dependent on the side chain structure rather than on the spacer. Therefore collisions between adjacent side chains cause large η values in FLCPs rather than hindrance to side chain gyration due to the spacer group. By combining FLCPs with ITO-coated plastic substrates, a large area matrix driven display has been made. Although an improvement in switching time is still needed, a flexible and lightweight display like a sheet of paper will be available in the near future.     

A New Synthesis of 3-Deoxy-D-Manno-Octulosonic Acid (KDO) From D-Mannose Via Condensation of Dioxene

Abstract

The title compound has long been known as an essential component of lipopolysaccharides (LPS) and capsular polysaccharides which exist in the outer membrane of Gram-negative bacteria. KDO has attracted additional attention as a consequence of some remarkable discoveries; i) mutants unable to produce KDO are non-viable.¹ ii) KDO is not present in mammalian cells,² iii) the 2-deoxy analog of β-KDO represents a new class of synthetic antimicrobial agent.^3,4 Although syntheses of KDO have been reported by many groups,^5–8 a more efficient synthetic method endowed with the potential for synthesis of not only complex glycoconjugates but also biologically significant analogs is required.     

Direct Synthesis of 1,4‐Diols from Alkenes by Iron‐Catalyzed Aerobic Hydration and C?H Hydroxylation

Various 1,4‐diols are easily accessible from alkenes through iron‐catalyzed aerobic hydration. The reaction system consists of a user‐friendly iron phthalocyanine complex, sodium borohydride, and molecular oxygen. Furthermore, the effect of additional ligands on the iron complex was examined for a model reaction. The second hydroxy group is installed by direct C(sp³) H oxygenation, which is based on a [1,5] hydrogen shift process of a transient alkoxy radical that is formed by formal hydration of the olefin.     

Regulation of the Cyanobacterial Circadian Clock by Electrochemically Controlled Extracellular Electron Transfer

There is growing awareness that circadian clocks are closely related to the intracellular redox state across a range of species. As the redox state is determined by the exchange of the redox species, electrochemically controlled extracellular electron transfer (EC‐EET), a process in which intracellular electrons are exchanged with extracellular electrodes, is a promising approach for the external regulation of circadian clocks. Herein, we discuss whether the circadian clock can be regulated by EC‐EET using the cyanobacterium Synechococcus elongatus PCC7942 as a model system. In vivo monitoring of chlorophyll fluorescence revealed that the redox state of the plastoquionone pool could be controlled with EC‐EET by simply changing the electrode potential. As a result, the endogenous circadian clock of S. elongatus cells was successfully entrained through periodically modulated EC‐EET by emulating the natural light/dark cycle, even under constant illumination conditions. This is the first example of regulating the biological clock by electrochemistry.     

Neomacrophorin I,II, and III,novel drimenyl cyclohexanes with hydroxylated butanoates from Trichoderma sp. 1212-03

Neomacrophorins I (1), II (2), and III (3) were isolated from the culture broth of Trichoderma sp. 1212-03, which was collected at Shirakami Mountainous area in Japan. Structural analyses disclosed that these resemble known macrophorins but possess axial-hydroxy group at C3 as well as different side chains at C7′. These are diastereomeric forms of macrophorins for 5′,6′-epoxide functionality. The NMR analyses suggested their relative configurational relationship between the C1–C15 drimene and C1′–C7′ epoxyquinone moieties. ECD spectral discussions verified them particularly for C5′,C6′-epoxyquinone (1), C5′,C6′-epoxysemiquinone (2 and 3), and 2″,3″-dihydroxybutanoate moiety in 1 and 2. The configuration of C3″-stereocenter of 3 was determined by chiral GC–MS after converting into methyl (S)-3″-hydroxybutanoate by basic of 3 methanolysis. Biological assays disclosed that 1 induces hyphal branching of Cochliobolus miyabeanus as well as cytotoxicity against human colorectal cancer COLO 201.     

Hyperpolarized 13C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study

Hyperpolarized [1-¹³C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1-¹³C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1-¹³C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1-¹³C]fumarate at sufficient ¹³C polarization for ex vivo detection of hyperpolarized ¹³C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo ¹³C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.