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PubMed2003

[Lipid rafts and their analytical methods].

Microdomains in cell membranes consist of caveolae and lipid rafts, in which cholesterol, glycolipids, and sphingomyelin are concentrated. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are unstable, being dynamically produced and degraded. In lipid rafts, flotillin is assumed to be one of the specifically located proteins. Since microdomains contain several signaling molecules, such as transmembrane receptors, they have an important role in receptor-medicated signal transduction. Caveolae or lipid rafts are known to be resistant to non-ionic detergents, such as Triton X-100. Because of this property, they are separated as the detergent-resistant membranes when the Triton X-100-treated cell lysate is subjected to sucrose gradient centrifugation. On the other hand, cholesterol is an essential molecule to maintain microdomain structure. When the cells are treated with cholesterol removing agents, such as methyl-beta-cyclodextrin and filipin, the microdomain in cell membranes is disrupted. Thus, the cholesterol removing agents are utilized to determine whether the microdomain is involved in certain cellular/physiological responses. Recently, green fluorescent protein-tagged protein is used to analyze the localization of the protein in lipid rafts in intact cells. Research on lipid rafts will be helpful for understanding the detailed mechanism of signal transduction and to clarify the molecular basis of several diseases.

Caveolin 1↗
PubMed2001

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis.

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.

Aequorin↗
PubMed2000

Three-dimensional numerical modeling for global cochlear dynamics.

A hybrid analytical-numerical model using Galerkin approximation to variational equations has been developed for predicting global cochlear responses. The formulation provides a flexible framework capable of incorporating morphologically based mechanical models of the cochlear partition and realistic geometry. The framework is applied for a simplified model with an emphasis on application of hybrid methods for three-dimensional modeling. The resulting formulation is modular, where matrices representing fluid and cochlear partition are constructed independently. Computational cost is reduced using two methods, a modal-finite-element method and a boundary element-finite-element method. The first uses a cross-mode expansion of fluid pressure (2.5D model) and the second uses a waveguide Green's-function-based boundary element method (BEM). A novel wave number approach to the boundary element formulation for interior problem results in efficient computation of the finite-element matrix. For the two methods a convergence study is undertaken using a simplified passive structural model of cochlear partition. It is shown that basilar membrane velocity close to best place is influenced by fluid and structural discretization. Cochlear duct pressure fields are also shown demonstrating the 3D nature of pressure near best place.

Auditory Perception↗
PubMed1991

Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography.

A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simulataneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 microns C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 microL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.

Anticonvulsants↗
PubMed2000

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).

Binding, Competitive↗
PubMedDate not supplied

Determination of spinosad in vegetables and fruits by high-performance liquid chromatography with UV and mass spectrometric detection after gel permeation chromatography and solid-phase extraction cleanup on a 2-layered column.

A simple and reliable method was developed for the determination of spinosyns A and D, the active ingredients of spinosad, in vegetables and fruits, by high-performance liquid chromatography with UV detection (HPLC-UV) and confirmation by mass spectrometry (MS); the method uses selected gel permeation chromatography (GPC) and a 2-layered column for solid-phase extraction system. An aliquot of the crude sample extract obtained by acetonitrile extraction is loaded into the GPC system. The fraction containing spinosyns A and D is selectively collected and loaded directly onto a 2-layered column consisting of graphitized carbon (upper layer) and cyclohexyl-bonded silica gel (lower layer). After the column is washed with the GPC mobile phase acetone-cyclohexane (3 + 7), the column is eluted with acetonitrile containing 2% triethylamine. The eluate is used for HPLC-UV/MS analysis. Average recoveries from fortified cabbage, green perilla, fig, and strawberry at analyte concentrations of 0.05 and 0.25 microg/g were >85%, and the relative standard deviations were <9%. The detection limits for spinosyns A and D in green perilla were 0.005 microg/g by UV detection and 0.001 microg/g by MS detection.

Chromatography, Gel↗
PubMed1996

Tributyltin distribution in the coastal environment of Peninsular Malaysia.

The occurrence of tributyltin (TBT) is reported in the coastal waters of a few selected sites in Peninsular Malaysia. Water, bivalves and sediment samples collected were analysed specifically for TBT using sensitive analytical methods which involved a solvent extraction procedure with appropriate clean-up followed by graphite furnace atomic absorption spectrometric measurements. The levels of TBT in the seawater in unexposed areas were found in the range from <3.4 to 20 ng litre(-1) as compared to coastal areas with high boat and ship activities where TBT levels in seawater were generally above 30 ng litre(-1), with the highest level found at 281.8 ng litre(-1). TBT levels in the tissues of random cockle and soft-shell clam samples from local markets were found in the range from <0.5 to 3.7 ng g(-1) wet weight. The levels of TBT found in green mussel samples both from the market (23.5 ng g(-1) wet weight) and those from a mussel farm (14.2 ng g(-1) wet weight) indicate slight accumulation of TBT. In sediments, TBT levels were found ranging from <0.7 ng g(-1) dry weight in unexposed coastal sites to as high as 216.5 ng g(-1) dry weight for a site within a port area.

Journal Article↗
PubMed2006

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing.

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.

Journal Article↗
PubMed2000

Compatibility and stability of linezolid injection admixed with three quinolone antibiotics.

OBJECTIVE: To evaluate the physical compatibility and chemical stability of linezolid 200 mg/100 mL admixed with ciprofloxacin 400 mg, ofloxacin 400 mg, and levofloxacin 500 mg for seven days at 4 and 23 degrees C. METHODS: The test samples were prepared by adding the required amount of the quinolone antibiotic to bags of linezolid injection. Evaluations for physical and chemical stability were performed initially and after one, three, five, and seven days of storage at temperatures of 4 and 23 degrees C. Physical stability was assessed using visual observation in normal light and using a high-intensity monodirectional light beam. In addition, turbidity and particle content were measured electronically. Chemical stability of the drugs was evaluated by using stability-indicating HPLC analytical techniques. RESULTS: The linezolid admixtures with levofloxacin and ofloxacin were clear and pale yellow when viewed in normal fluorescent room light, and slightly hazy with a green cast when viewed using a Tyndall beam. Measured turbidity and particulate content were low and exhibited little change. HPLC analysis found no loss of the drugs in any sample stored at either temperature throughout the study. The linezolid admixtures with ciprofloxacin stored at room temperature (23 degrees C) were clear and nearly colorless in normal room light and when viewed using a Tyndall beam. They exhibited little or no change in measured turbidity or particulate content during the study period. HPLC analysis found no loss of either drug in seven days. However, the refrigerated samples were only compatible for 24 hours and developed a gross white precipitate thereafter. CONCLUSIONS: Admixtures of linezolid 200 mg/100 mL with levofloxacin 500 mg and with ofloxacin 400 mg were physically compatible and chemically stable for at least seven days stored at 4 and 23 degrees C. Admixtures of linezolid with ciprofloxacin 400 mg were compatible and stable for seven days at 23 degrees C, but ciprofloxacin precipitation occurred after 24 hours stored under refrigeration. Linezolid/ciprofloxacin admixtures should not be stored under refrigeration.

Acetamides↗
PubMed1993

Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting.

OBJECTIVE: To determine the applicability of flow cytometry to assess human sperm acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated overnight for the development of spontaneous acrosome reaction or exposed to the calcium ionophore A23187 (5 microM) for 3 hours for induction of the acrosome reaction. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: The spermatozoa were stained with fluorescein isothiocyanate (FITC)-labeled pea agglutinin. The labeled samples were assessed visually and also subjected to analytic flow cytometry and fluorescence-activated cell sorting. MAIN OUTCOME MEASURES: Acrosome reaction assessed visually and by flow cytometry. RESULTS: Flow cytometric analysis showed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after overnight incubation or exposure to A23187, suggesting loss of fluorescence, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between the methods was small (2.49%), the difference between the two methods for each individual sample can vary between -24% to +29%. When the sperm cells were subjected to cell sorting based on green fluorescence intensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%). CONCLUSION: These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytometry is more objective and less time consuming when many samples are assessed at the same time, the visual method remains a useful and practical procedure in the clinical andrology laboratory.

Acrosome↗
PubMedDate not supplied

Determination of malachite green and its metabolite, leucomalachite green, in catfish (Ictalurus punctatus) tissue by liquid chromatography with visible detection.

To determine residues of malachite green (MG) and its metabolite, leucomalachite green (LMG), in catfish tissue, analytes are extracted with acetonitrile-buffer and the extract is partitioned into methylene chloride. Final cleanup and isolation are performed on neutral alumina solid-phase extraction (SPE) and propylsulfonic acid cation-exchange SPE columns before analysis by liquid chromatography with visible detection. PbO2 postcolumn oxidation is performed by isocratic elution with a buffered mobile phase from a cyano column. Recoveries and relative standard deviations (RSDs) from fortified catfish tissues were 72.9% (RSD, 1.92%; 23 ppb), 75.5% (RSD, 6.85%; 11 ppb), and 69.6% (RSD, 6.93%; 5.7 ppb) for MG and 87.4% (RSD, 2.92%; 21 ppb), 88.1% (RSD, 5.94%; 10 ppb), and 82.6% (RSD, 11.5%; 5.3 ppb) for LMG. The method was applied to MG-incurred catfish at depuration times of 0, 2, 4, 8, and 24 h. Average levels of residual MG and LMG in the 24 h depuration catfish tissue were 73.4 and 289 ppb, respectively.

Animals↗
PubMed2004

A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase.

BACKGROUND: Most of the polyamines circulating in blood are spermidine (Spd) and spermine (Spm) with only trace amounts of putrescine (Put), and they are mainly localized in erythrocytes. We developed a simple and sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase (OSPO). The method is based on the unique substrate specificity of OSPO, which is active toward Spd and Spm, but not toward diamines such as Put and cadaverine and monoamines such as histamine. METHODS: The polyamines, which were purified from packed erythrocytes by weak cation-exchange chromatography, were incubated with OSPO at 37 degrees C for 15 min. In the presence of the H(2)O(2) produced by this polyamine oxidase reaction and a new chromogen, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine sodium salt (DA-64), peroxidase (POD) catalyzes the formation of N-[4-[[4-(dimethylamino)phenyl]imino]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium chloride (Bindschedler's Green) having an absorption maximum at 727 nm. RESULTS: The detection limit was 0.2 microM/l for packed erythrocytes. The within-run and between-run precisions (coefficient of variation, CVs) were 5.6-15.2% and 6.5-16.4%, respectively. Analytical recoveries were 93.3-97.4%. Polyamine values obtained by this assay correlated well with those obtained by an HPLC (y=0.948x + 1.912; r=0.944; n=46). CONCLUSIONS: This colorimetric assay is simple and highly sensitive and practical for clinical use.

Adult↗
PubMed2006

Multivariate analysis for 1H-NMR spectra of two hundred kinds of tea in the world.

NMR measurements coupled with pattern-recognition analysis offer a powerful mixture-analysis tool for latent-feature extraction and sample classification. As fundamental applications of this analysis for mixtures, the 1H spectra of 176 kinds of green, black, oolong and other tea infusions were acquired by a 500 MHz NMR spectrometer. Each spectrum pattern was analyzed by a multivariate statistical pattern-recognition method where Principal Component Analysis (PCA) was used in combination with Soft Independent Modeling of Class Analogy (SIMCA). SIMCA effectively selected variables that contribute to tea categorization. The final PCA resulted in clear classification reflecting the fermentation and processing of each tea, and revealed marker variables that include catechin and theanine peaks.

Algorithms↗
PubMed2007

High-throughput liberation of water-soluble yeast content by irreversible electropermeation (HT-irEP).

The article describes a high-throughput method for the liberation of water-soluble cell contents by exploiting the phenomenon of irreversible membrane electropermeation (HT-irEP). The method is exemplified in recombinant proteins and plasmid liberation from yeast Saccharomyces cerevisiae on the detectable level. Obtained extracts are pure enough to be readily applied for further analytical analysis such as enzyme assay, PCR, and so on. From the same HT-irEP extract, one can measure activity of the target protein and perform amplification of the corresponding gene from the DNA vector by PCR for recombinant protein with intracellular expression. Therefore, the method is suitable for the high-throughput screening (HTS) of yeast libraries where extracellular expression of recombinant protein is problematic. The method can be easily automated and integrated into existing HTS systems.

Cell Membrane Permeability↗
PubMed1987

Enzyme resistant starch fractions and dietary fibre.

Starch fractions that are more or less enzyme resistant may behave like dietary fibre, both physiologically and analytically. Ungelatinized granules from potatoes, high amylose maize and green bananas are poorly digested. Starch made resistant to amylase due to new covalent bindings, formed at heat treatment or present in starch derivatives used as food additives, may also be more or less undigestible. "Resistant starch" present in bread and corn flakes is probably retrograded amylose. It is undigestible in the small intestine, but readily degraded by the large bowel microflora. Amylose-lipid complexes seem to be completely absorbed in spite of their resistance to amylase degradation in vitro. Since undigestible starch fractions behave physiologically like non-starch polysaccharides, they should be included in the dietary fibre concept. "Resistant starch" is analysed as glucose based fibre with all current methods except one, which includes an initial DMSO solubilization step.

Amylases↗
PubMed1999

Capillary electrophoresis and capillary electrochromatography of organic pollutants.

Capillary electrophoresis (CE) has a unique capability for separation of analytes of environmental concern, particularly those that are more polar and ionic, based on the complementary separation principle of electrophoresis. In the past few years, CE has been selectively used to analyze various classes of compounds having current or potential environmental relevance. This review outlines the current status of CE for the determination of environmental pollutants, based predominantly on research results published from the beginning of 1997 to early 1999. Covered are environmental pollutants of all types except pesticides and inorganics. Certain naturally produced toxins are also covered because of their significant impacts upon human health and the environment. CE methods, as with all methods, must be judged on their ability to provide approaches that are reliable, sensitive, selective, and rapid, while meeting "green chemistry" initiatives for pollution prevention. We also compare CE methods to benchmark environmental techniques involving gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high performance liquid chromatography (HPLC).

Animals↗
PubMed2006

Finite-difference calculation of the Green's function of a one-dimensional crystal: application to the Krönig-Penney potential.

We present a finite-difference scheme for computing the Green's function of a one-dimensional crystal. The method enables one to derive the band structure and the density of states of this type of structures, whatever the particular values of the potential energy. The technique also enables one to compute the influence of defects on the density of states and on the scattering of the eigenstates of the crystal. The technique is applied to the Krönig-Penney potential. In particular, we study the bound states of a square potential introduced in the crystal and their influence on the conductance of the system. We also determine the surface states induced by a termination of the Krönig-Penney potential. Our results turn out to be in excellent agreement with analytical expressions, which proves their validity and the versatility of the technique.

Journal Article↗
PubMed1984

Analysis of an Effective Antibiotic (Chaetomacin) Isolated from a Thermophilic Bacillus sp. Against Olive Green Mold.

Successful methods to control the damaging weed mold Chaetomium olivaceum (olive green mold) in mushroom beds are not known. An effective antibiotic (named chaetomacin) against C. olivaceum was isolated from a thermophilic Bacillus sp. This compound was shown to be an extremely potent and stable antibiotic, effective over a wide range of both pH (2 to 10) and temperature (-15 to 150 degrees C). Chaetomacin is soluble in most polar solvents and insoluble in nonpolar solvents. It is produced only at mesophilic temperatures and is also active against other Bacillus spp. and various eucaryotes, but it demonstrates no activity against gram-negative rods or gram-positive cocci. Final purification of chaetomacin was accomplished through thin-layer chromatography on silica gel analytical plates. Amino acid analysis revealed the antibiotic to be a peptide, acidic in nature. Examination of the literature reveals no other previously isolated antibiotics identical to chaetomacin.

Journal Article↗