We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.
Anilino Naphthalenesulfonates ; chemistry ; Enzyme Stability ; High-Throughput Screening Assays ; methods ; Hot Temperature ; Lipase ; metabolism ; Spectrometry, Fluorescence

This study examined the mechanism of action of a local anesthetic, lidocaine.HCl. Energy transfer between the surface fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, and the hydrophobic fluorescent probe, 1,3-di(1-pyrenyl) propane, was used to determine the effect of lidocaine.HCl on the thickness (D) of the synaptosomal plasma membrane vesicles (SPMV) isolated from the bovine cerebral cortex, and liposomes of the total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. The thickness (D) of the intact SPMV, SPMVTL and SPMVPL were 1.044+/-0.008, 0.914+/-0.005 and 0.890+/-0.003 (arbitrary units, n=5) at 37degrees C (pH 7.4), respectively. Lidocaine.HCl decreased the thickness of the neuronal and model membrane lipid bilayers in a dose-dependent manner with a significant decrease in the thickness, even at 0.1 mM. The decreasing effect of lidocaine.HCl on the membrane thickness might be responsible for some, but not all of its anesthetic action.
Anilino Naphthalenesulfonates ; Cell Membrane ; Cerebral Cortex ; Energy Transfer ; Lipid Bilayers ; Liposomes ; Membranes ; Neurons ; Phospholipids ; Propane

BACKGROUNDVolatile anesthetics (VAs) may affect varied and complex physiology processes by manipulating Ca(2+)-calmodulin (CaM). However, the detailed mechanism about the action of VAs on CaM has not been elucidated. This study was undertaken to examine the effects of VAs on the conformational change, hydrophobic site, and downstream signaling pathway of CaM, to explore the possible mechanism of anesthetic action of VAs.

METHODSReal-time second-harmonic generation (SHG) was performed to monitor the conformational change of CaM in the presence of VAs, each plus 100 µmol/L Ca(2+). A hydrophobic fluorescence indicator, 8-anilinonaphthalene-1-sulfonate (ANS), was utilized to define whether the VAs would interact with CaM at the hydrophobic site or not. High-performance liquid chromatography (HPLC) was carried out to analyze the activity of CaM-dependent phosphodiesterase (PDE1) in the presence of VAs. The VAs studied were ether, enflurane, isoflurane, and sevoflurane, with their aqueous concentrations 7.6, 9.5, 11.4 mmol/L; 0.42, 0.52, 0.62 mmol/L; 0.25, 0.31, 0.37 mmol/L and 0.47, 0.59, 0.71 mmol/L respectively, each were equivalent to their 0.8, 1.0 and 1.2 concentration for 50% of maximal effect (EC50) for general anesthesia.

RESULTSThe second-harmonic radiation of CaM in the presence of Ca(2+) was largely inhibited by the VAs. The fluorescence intensity of ANS, generated by binding of Ca(2+) to CaM, was reversed by the VAs. HPLC results also showed that AMP, the product of the hydrolysis of cAMP by CaM-dependent PDE1, was reduced by the VAs.

CONCLUSIONSOur findings demonstrate that the above VAs interact with the hydrophobic core of Ca(2+)-CaM and the interaction results in the inhibition of the conformational change and activity of CaM. This in vitro study may provide us insight into the possible mechanism of anesthetic action of VAs in vivo.

Adenosine Monophosphate ; analysis ; Anesthetics, Inhalation ; pharmacology ; Anilino Naphthalenesulfonates ; Calmodulin ; antagonists & inhibitors ; chemistry ; physiology ; Cyclic Nucleotide Phosphodiesterases, Type 1 ; analysis ; Fluorescence ; Humans ; Hydrophobic and Hydrophilic Interactions

OBJECTIVES: Deficit schizophrenia (DS) constitutes a disease separate from non-deficit schizophrenia (NDS). The aim of the current study was to compare the quantitative EEG and low resolution electromagnetic tomography (LORETA) imaging between DS and NDS. METHODS: This study was performed by 32 channels EEG for 42 schizophrenia patients who we categorized into DS and NDS using proxy instrument deficit syndrome (PDS). We performed the absolute power spectral analyses for delta, theta, alpha, low beta and high beta activities. We compared power spectrum between two groups using Independent t-test. Partial correlation test was performed with clinical parameters. Standardized LORETA (sLORETA) was used for comparison of cortical activity, and statistical nonparametric mapping (SnPM) was applied for the statistical analysis. RESULTS: DS showed significantly increased delta and theta absolute power in fontal and parietal region compared with NDS (p<0.05). Power spectrum showed significant correlation with 'anergia' and 'hostility/suspiciousness' subscale of brief psychiatric rating scale (BPRS)(p<0.05). sLORETA found out the source region (anterior cingulate cortex/limbic part) that delta activity was significantly increased in DS (p=0.042). CONCLUSIONS: DS showed different cortical activity compared with NDS. Our results may suggest QEEG and LORETA could be the marker in differentiating between DS and NDS.
Brief Psychiatric Rating Scale ; Electroencephalography ; Humans ; Magnets ; Naphthalenesulfonates ; Proxy ; Schizophrenia

Because the red and bright color of corolla is the main indicator for the quality assessment of good safflower,the dyed safflower is sometimes found at the herbal market,what is influence on this herb quality and efficacy. A total of 127 safflower samples was therefore collected from different cultivating areas and herbal markets in China to develop a rapid method to identify the dyed safflower. Near-infrared spectroscopy(NIRS) combined with characteristic identification,high performance liquid chromatography(HPLC),principal component analysis(PCA) and partial least squares regression analysis(PLS) were employed to differentiate safflower from dyed safflower samples,and further quantify the levels of the 6 dyes,i.e. tartrazine,carmine,sunset yellow,azorubine,acid red 73 and orange Ⅱ in the dyed safflower. The results indicated that the 50 safflower samples and 77 dyed safflower samples were located at different regions in PCA cluster diagram by NIR spectra. Tartrazine,carmineand and sunset yellow were found in the 77 dyed safflower samples with the amounts of 0. 60-3. 66,0. 11-1. 37,0. 10-0. 71 mg·g-1,respectively. It indicated that the three dyes were the common and main dyes in the dyed safflower. However,azorubine,acid red 73 and orange Ⅱ were not detected in all herb samples. A total of 62 dyed safflower samples were chosen as calibration samples to develop the model for estimating the amount of dyes in dyed safflower. The estimating accuracy was verified by another 15 dyed safflower samples. The values of tartrazine,carmine and sunset yellow in dyed safflower samples were compared between the NIRS and HPLC methods. Each value of mean absolute difference(MAD) was less than 5%. The correlation coefficients of tartrazine,carmineand and sunset yellow were 0. 970,0. 975,0. 971,respectively. It indicated the data quantified by NIRS and HPLC were consistence. It is concluded that NIRS can not only differentiate safflower from dyed safflower,but also quantify the amount of the dyes. NIRS is suitable for rapidly identify the quality of safflower.
Azo Compounds ; Benzenesulfonates ; Carmine ; Carthamus tinctorius ; chemistry ; China ; Coloring Agents ; analysis ; Naphthalenesulfonates ; Spectroscopy, Near-Infrared ; Tartrazine

OBJECTIVEA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HIV-1.

METHODSRT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The performance of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR(qRT-PCR).

RESULTSThe RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100%, respectively,in comparison with those of qRT-PCR.

CONCLUSIONRT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.

HIV-1 ; isolation & purification ; Naphthalenesulfonates ; Nucleic Acid Amplification Techniques ; methods ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Reverse Transcription ; Sensitivity and Specificity

OBJECTIVETo develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).

METHODSThe method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.

RESULTSA positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.

CONCLUSIONThe established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.

Colorimetry ; Coloring Agents ; chemistry ; DNA Primers ; Enterovirus ; isolation & purification ; Hand, Foot and Mouth Disease ; virology ; Humans ; Indicators and Reagents ; chemistry ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; Sensitivity and Specificity

To determine the optimal media conditions for the detection of the extracellular cellulase activity in Ganoderma neo-japonicum, we varied three media conditions: dye reagent, pH, and temperature. We evaluated the use of four dyes, Congo red, phenol red, remazol brilliant blue, and trypan blue. To observe the effect of pH on the chromogenic reaction, we tested media ranging from 4.5 to 8.0. To research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to 35degrees C. On the whole, the best protocol called for Ganoderma neo-japonicum transfer onto media containing Congo red with a pH of 7.0, followed by incubation at 25degrees C for 5 days. Our results will be useful to researchers who study extracellular enzyme activity in Ganoderma neo-japonicum.
Benzenesulfonates ; Cellulase ; Coloring Agents ; Congo Red ; Diminazene ; Fungi ; Ganoderma ; Hydrogen-Ion Concentration ; Phenolsulfonphthalein ; Trypan Blue

To determine the optimal medium conditions for the detection of the cellulolytic activity in Ganoderma lucidum, we varied three media conditions: dye reagent, pH, and temperature. First, we evaluated the use of four dyes, Congo Red, Phenol Red, Remazol Brilliant Blue, and Trypan Blue. To observe the effect of pH on the chromogenic reaction, we also made and tested various media spanning acidic and alkaline pHs, ranging from 4.5 to 8.0. Furthermore, in order to research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to 35degrees C. On the whole, the best protocol called for Ganoderma lucidum transfer onto media containing Congo red with pH adjusted to 7.0, followed by incubation at 25degrees C for 5 days. Our results will be useful to researchers who aim to study extracellular enzyme activity in Ganoderma lucidum.
Benzenesulfonates ; Coloring Agents ; Congo Red ; Diminazene ; Fungi ; Ganoderma ; Hydrogen-Ion Concentration ; Phenolsulfonphthalein ; Reishi ; Trypan Blue

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Animals ; Colorimetry ; methods ; DNA Primers ; genetics ; Electrophoresis, Agar Gel ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; methods ; Orthomyxoviridae Infections ; diagnosis ; veterinary ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine ; Swine Diseases ; diagnosis ; virology ; Temperature