Detection of dopamine levels using a polydiacetylene liposomal aequorin bioluminescent device with octadecylboronic acid.

The development of an easy-to-use and rapid method for the determination of dopamine levels is desirable for the diagnosis of neurological conditions, such as Parkinson's disease, which are characterized by low levels of dopamine. Herein, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) containing octadecylboronic acid (OBA) as a recognition material (PLABD-OBA) was prepared for the determination of dopamine concentrations in aqueous solution. The bioluminescent signals of the photoprotein aequorin in PLABD-OBA increased according to increasing dopamine concentrations. The calibration curve showed good linearity over a dopamine concentration range of 70-700 µM (r = 0.918), with a detection limit of 7.5 µM. The addition of other catecholamines to the PLABD-OBA resulted in low bioluminescent signals of aequorin. Because the physiological levels of dopamine are generally 0.001-1.0 µM, this system had insufficient sensitivity for the clinical monitoring of dopamine levels. However, the PLABD-OBA developed herein is an easy-to-use and rapid analytical method that is specific for dopamine.

Spectroscopic study of cyclen-based 19F NMR probe for detection of hydrogen sulfide.

A cyclen-based 19F NMR probe (F-cyclen) for hydrogen sulfide (H2S) has been prepared and evaluated for its complex formation ability with Cu2+ ions and responsivity to H2S. F-Cyclen was readily synthesized by reacting cyclen with 4-(trifluoromethyl)benzyl bromide. Visible absorption spectrophotometry showed that, same as the original cyclen, F-cyclen formed a 1:1 complex with Cu2+ ions. The 19F NMR signal of F-cyclen at 16.5 ppm gradually decreased in intensity with increasing CuCl2 concentration, with trifluoromethane sulfonic acid sodium salt (TFMSNa) used as an internal standard (0 ppm). When the Cu2+-F-cyclen complex was subjected to an increasing concentration of Na2S (as H2S donor), its corresponding 19F NMR signal of F-cyclen at 16.5 ppm gradually increased in intensity. The regression curve between the 19F NMR signal intensity ratio of F-cyclen to TFMSNa and Na2S concentration showed good linearity (r = 0.986) over the Na2S concentration range of 25-150 µM.

Application of Molecularly Imprinted Polymer-modified Potentiometric Sensor for Quantitative Determination of Histamine in Serum.

A molecularly imprinted polymer-modified potentiometric histamine (HIS) sensor was prepared and used for quantitative determination of HIS in bovine serum. The calibration curve using the potential responses measured in 1 × 10-3 mol L-1 phosphate buffer (pH 7.4) showed good linearity in the HIS concentration range of 3 × 10-4 to 1 × 10-2 mol L-1 (r = 0.92), with a detection limit of 1.6 × 10-4 mol L-1. In bovine serum samples, the HIS sensor showed good recovery values of 91 - 104%. Therefore, this HIS sensor successfully determined the HIS concentration in bovine serum samples.

Antihypertensive constituents in Sanoshashinto.

It has been reported that Sanoshashinto (SanHuangXieXinTang, ), which is composed of Rhei Rhizoma, Scutellariae Radix, and Coptidis Rhizoma, exhibits vasorelaxant effects in vitro and lowers blood pressure of patients. Based on this discovery, in this study, a mixture containing those three materials and combinations of them were extracted with methanol, and the extracts were fractionated into different parts. Effects of all extracts and fractions on high concentration of potassium chloride (High K+)- or noradrenaline (NA)-induced contractions of isolated rat aortic rings or helical strips were examined. Qualitative and quantitative HPLC analyses of the extracts and the fractions revealed that the contents of baicalin and berberine in Sanoshashinto methanol extract (SHXXTM) were higher than those of the other constituents. All pharmacological and HPLC data were analyzed by principal component analysis (PCA) software and the results indicated that baicalin, berberine, palmatine, baicalein, and wogonoside contributed significantly to the pharmacological activity. Furthermore, spontaneously hypertensive rats (SHRs) that were orally given SHXXTM or a baicalin-berberine combination showed significantly reduced increase in the rate of systolic blood pressure (SBP) compared to the control group. These findings suggested that Sanoshashinto has significant vasorelaxant effects in vitro and antihypertensive effects in vivo, and baicalin and berberine, which were the principal constituents of Scutellariae Radix and Coptidis Rhizoma, were the main antihypertensive constituents in Sanoshashinto. It was speculated that baicalin and berberine produced vasorelaxant effects by activating the NO/cGMP pathway and that the BKCa channel and the DAG/PKC/CPI-17 pathway were also involved.

Effects of mono- and dialkylglucosides on the characterisation and blood circulation of lipid nanoemulsions.

Aim: Effects of two cosurfactants, n-alkylglycosides with mono- or disaccharide groups - N-nonyl ß-D-glucopyranoside (N-Glu) and N-decyl ß-D-maltoside (D-Mal) - were studied to the stability in saline solution, interaction with serum albumin, and blood circulation of the lipid nanoemulsion (LNE).Methods: The LNEs composed of soybean oil, phosphatidylcholine, and sodium palmitate were prepared without (Control-LNE) and with N-Glu or D-Mal (NG-LNE and DM-LNE, respectively).Results: In saline solution, NG-LNE exhibited a smaller droplet size than Control-LNE, while the size of DM-LNE was significantly increased compared with the other LNEs. The fluorescence resonance energy transfer method showed that the order of albumin interaction was DM-LNE > NG-LNE > Control-LNE. In vivo blood circulation in mice, showed greater fractions of both NG-LNE and DM-LNE remaining in blood over time compared with Control-LNE.Conclusions: The nature of high stability in saline solution and high affinity for serum albumin led to the prolonged circulation of LNE.

A Molecularly Imprinted Polymer-modified Potentiometric Sensor for the Detection of Glutathione.

Potentiometric glutathione (GSH) sensors were fabricated using a molecularly imprinted polymer prepared from GSH, methacrylic acid (MAA), and ethylene glycol dimethacrylate as the template molecule, functional monomer, and cross-linker, respectively. Five GSH sensors were prepared with different ratios of GSH to MAA. Their potential responses were measured in a GSH aqueous solution using Ag/AgCl as the reference electrode. A GSH sensor prepared with a GSH:MAA ratio of 2:32 had the best responsivity, while the sensor synthesized from a non-imprinted polymer prepared without GSH (NIP sensor) showed a potential response value of almost zero after the addition of GSH. The ratio of the potential responses of the GSH sensor to the NIP sensor was 8.21. Additionally, the GSH sensor had good linearity over a GSH concentration range of 1 × 10-5 to 2 × 10-4 mol L-1. The GSH sensor provided good quantification and high specificity for GSH and is expected to be applicable for easy and direct determinations of GSH.

Effects of Helicobacter pylori eradication on serum lipid levels.

The purpose was to clarify the effects of Helicobacter pylori (H. pylori) eradication on the changes in serum lipid levels by comparing subjects with and without continuous H. pylori infection. The study subjects were 774 individuals (males 536, females 238, mean age 52.6 years) who visited between April 2013 and March 2016 for annual medical checkups. Serum total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), and triglyceride levels, and LDLC/HDLC ratio were compared between the subjects with and without H. pylori infection, as well as those with H. pylori eradication subjects. The HDLC level in the H. pylori-positive group was significantly lower as compared to the H. pylori-negative group. The serum level of HDLC in subjects with successful eradication of H. pylori tended to be higher, while the serum levels of total cholesterol, LDLC, and triglycerides tended to be lower in comparison to subjects with continuous H. pylori infection. In addition, the LDLC/HDLC ratio in the H. pylori-positive group was significantly higher than that in the H. pylori-negative group, and successful H. pylori eradication tended to reduce that ratio. In conclusion, successful eradication of H. pylori may have favorable effects on lipid metabolism.

Combination of 1H nuclear magnetic resonance spectroscopy and principal component analysis to evaluate the lipid fluidity of flutamide-encapsulated lipid nanoemulsions.

The lipid fluidity of various lipid nanoemulsions (LNEs) without and with flutamide (FT) and containing one of two neutral lipids, one of four phosphatidylcholines as a surfactant, and sodium palmitate as a cosurfactant was investigated by the combination of 1H nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA). In the 1H NMR spectra, the peaks from the methylene groups of the neutral lipids and surfactants for all LNE preparations showed downfield shifts with increasing temperature from 20 to 60 °C. PCA was applied to the 1H NMR spectral data obtained for the LNEs. The PCA resulted in a model in which the first two principal components (PCs) extracted 88% of the total spectral variation; the first PC (PC-1) axis and second PC (PC-2) axis accounted for 73 and 15%, respectively, of the total spectral variation. The Score-1 values for PC-1 plotted against temperature revealed the existence of two clusters, which were defined by the neutral lipid of the LNE preparations. Meanwhile, the Score-2 values decreased with rising temperature and reflected the increase in lipid fluidity of each LNE preparation, consistent with fluorescence anisotropy measurements. In addition, the changes of Score-2 values with temperature for LNE preparations with FT were smaller than those for LNE preparations without FT. This indicates that FT encapsulated in LNE particles markedly suppressed the increase in lipid fluidity of LNE particles with rising temperature. Thus, PCA of 1H NMR spectra will become a powerful tool to analyze the lipid fluidity of lipid nanoparticles. Graphical abstract ᅟ.

Thermal Behavior of 19F Nuclear Magnetic Resonance Signal of 19F-Containing Compound in Lipid Nano-Emulsion for Potential Tumor Diagnosis.

We developed carriers of a 19F magnetic resonance imaging (19F MRI) agent, capable of responding to the temperature difference for cancer diagnosis. The carriers were based on high melting point (mp) neutral lipids, namely, tripalmitin (TPT) and tristearin (TSR) and triarachidin (TAC). Lipid nano-emulsions (LNEs) containing a fluorine compound, i.e., a modified α-tocopherol (19F-TP), were respectively prepared as TPT-LNE, TSR-LNE, TAC-LNE1, and TAC-LNE2 and studied by 19F NMR spectroscopy. In LNE prepared with soybean oil as a control, the full width at half maximum (FWHM) values of the 19F NMR signal of 19F-TP remained constant at 25, 37, and 42°C, while those of the LNEs prepared from a neutral lipid with a high mp showed a sharp decrease between 25 and 37°C. The magnitude of the decrease followed the order: TPT-LNE < TSR-LNE < TAC-LNE1. However, TAC-LNE2, for which the amount of encapsulated 19F-TP was one third less than that of TAC-LNE1, showed a sharp decline in the FWHM between 37 and 42°C. To examine these changes, the 19F spin-lattice (T1) and spin-spin (T2) relaxation times of 19F-TP were measured. TAC-LNE2 in particular showed a substantial change in its T2 value between 37 and 42°C compared with the change of its T1 value. This result was attributed to activation of the molecular motion of 19F-TP in TAC-LNE2 from 37 to 42°C. Thus, TAC-LNE showed potential for use as a carrier for cancer diagnosis using 19F MRI.

Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds.

In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 µmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 µmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.

19F Nuclear Magnetic Resonance Spectrometric Determination of the Partition Coefficients of Flutamide and Nilutamide (Antiprostate Cancer Drugs) in a Lipid Nano-Emulsion and Prediction of Its Encapsulation Efficiency for the Drugs.

To design a useful lipid drug carrier having a high encapsulation efficiency (EE%) for the antiprostate cancer drugs flutamide (FT) and nilutamide (NT), a lipid nano-emulsion (LNE) was prepared with soybean oil (SO), phosphatidylcholine (PC), and sodium palmitate, and the partition coefficients (K ps) of the drugs for the LNE were determined by 19F nuclear magnetic resonance (NMR) spectrometry. The 19F NMR signal of the trifluoromethyl group of both drugs showed a downfield shift from an internal standard (trifluoroethanol) and broadening according to the increase in the lipid concentration due to their interaction with LNE particles. The difference in the chemical shift (Δδ) of each drug caused by the addition of LNE was measured under different amounts of LNE, and the K p values were calculated from the Δδ values. The results showed that FT has higher lipophilicity than NT. The total lipid concentration (SO + PC) required to encapsulate each drug into LNE with an EE% of more than 95% was calculated from the K p values as 93.3 and 189.9 mmol/L for FT and NT, respectively. For an LNE prepared with the total lipid concentration of 215 mmol/L, the predicted EE% values were 98 and 96% for FT and NT, respectively, while the experimental EE% values determined by a centrifugation method were approximately 99% for both drugs. Thus, the 19F NMR spectrometric method is a useful technique to obtain the K p values of fluorinated drugs and thereby predict the theoretical lipid concentrations and prepare LNEs with high EE% values.

Behavioral analyses of wind-evoked escape of the cricket, Gryllodes sigillatus.

The wind-evoked escape behavior of the cricket Gryllodes sigillatus was investigated using an air puff stimulus. A high velocity air puff elicited the escape behavior in many crickets. The crickets tended to escape away from the stimulus source, but the direction was not accurately oriented 180 degrees from the stimulus. After bilateral cercal ablation, only a few crickets showed wind-evoked escape behavior, and their response rates did not increase even 19 days after ablation. Therefore, information on air motion detected by cercal filiform hairs is essential for triggering wind-evoked behavior. After unilateral cercal ablation, the 81.3% response rate of intact crickets decreased to 16.5%, that is, it decreased to almost 20% that of intact crickets. One week after unilateral cercal ablation, the response rate recovered to more than 60% that of intact crickets. However, the accuracy rate of the escape direction of G. sigillatus showed no change even immediately after the unilateral cercal ablation. Therefore, both cerci are not necessarily required to determine the escape direction. The behavioral characteristics of wind-evoked escape of G. sigillatus are compared with those of another species of cricket, Gryllus bimaculatus. The two species of cricket employ different strategies for wind-evoked escape.