Invasive epithelial cells show more fast plasma membrane movements than related or parental non-invasive cells.

Fast plasma membrane movements (FPMM) are involved in ruffling, blebbing, fast shape change, and fast translocation. A simple method for the quantification of FPMM was used to study the relation between FPMM and invasive capacity in five pairs of invasive and noninvasive variants from four different epithelial cell types. The human mammary cell line MCF-7/6, the ras-transformed dog kidney cell line ras-MDCK, the ras-transformed mouse mammary gland cell lines NM9-ras-12 and NM-f-ras-TD, and spontaneously transformed late passage mouse lens explant MLE cells, all of which were invasive in vitro, showed more FPMM in our measurements and displayed more ruffling activity on time-lapse video films than the related or parental MCF-7/AZ, MDCK-3, NM9, and NM-f cell lines and early passage MLE cells, none of which were invasive. Interestingly, induction of invasive capacity in MCF-7/AZ cells by retinoic acid was accompanied by an increase in FPMM, but speed of translocation was not increased. Together these observations support the hypothesis that a certain level of FPMM is a prerequisite for invasive capacity.     

Simple and sensitive method for quantification of low tobramycin concentrations in human plasma using HPLC-MS/MS

After oral administration of tobramycin, as part of selective decontamination of the digestive tract (SDD) in critically ill patients, absorption of tobramycin from the gut into the blood may take place. To quantify low concentrations of tobramycin in human plasma, we developed and validated a simple (sample pre-treatment consisting of protein precipitation with acetonitrile using 200microl plasma), rapid (runtime 3min using a Pathfinder MR reversed-phase column) and sensitive (concentration range of 0.05-1.0mg/l using MS/MS detection) method.     

Simple fluorimetric method for quantification of sialic acids in glycoproteins

A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0 degrees C for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180 microg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5 microg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.     

Simple screening method for improving membrane protein thermostability

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.     

Simple, reliable and fast spectrofluorometric method for determination of plasma Verteporfin (Visudyne) levels during photodynamic therapy for choroidal neovascularization.

Photodynamic therapy with Verteporfin, a potent photosensitizer dye, is a very effective treatment for age related macular degeneration due to choroidal neovascularization. Photodynamic therapy offers the potential for selective tissue injury in part attributable to preferential localization of Verteporfin, administrated by intravenous infusion, to the choroidal neovascularization complex and irradiation of the complex with non-laser thermal light at 690 nm resulting in at least temporary thrombosis and vessel closure. Verteporfin is a benzoporphyrin derivative monoacid ring A formulated as a unilamellar liposome. In the blood Verteporfin is associated with lipoprotein fractions and is rapidly cleared via a receptor-mediated uptake mechanism due the high expression of LDL receptors in neovascular tissues. Verteporfin was undetectable in plasma 24 hr after infusion of the recommended dose: 6 mg/m2 of body surface area. The main side effect is photosensitivity of skin which is usually short-lived (24-48 hr) with a low incidence (2.3%). As skin photosensitivity depends on circulating rather than tissue drug levels, we investigate the possibility of developing a simple, fast and reliable spectrofluorometric method to measure plasma Verteporfin levels. Fluorescence emission spectrum (550-750 nm) of 1:10 saline diluted plasma with lambda exc=430 nm showed a characteristic emission peak at 692 nm, the height being proportional to the Verteporfin levels. The sensitivity is around 100 ng/ml and the pharmacokinetics of Verteporfin has been studied from 0 to 5 hr after infusion in six patients older than 65 years with age-related macular degeneration.     

Improved HPLC method for total plasma homocysteine detection and quantification

Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R=0.999), range (<2-100 microM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B(6), on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.     

Simple and rapid high-performance liquid chromatographic method for the quantification of 3-methylhistidine

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.     

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Sample preparation method for plasma membrane proteome analysis

The preparation of plasma membrane (PM) proteome samples is seriously difficult and time-consuming, owing to their profound hydrophobicity and low abundance. We have developed an efficient PM sample preparation method using Ultracentrifugation with Percoll and an aqueous two-phase extraction. The developed method was rapid (3h) and provided high purities (26-fold of cell lysate) with a high yield (2.6% of whole cell lysate proteins). This method is especially useful for PM proteome studies using 2D gel electrophoresis.     

A fast method for the quantification of methylamine in fermentation broths by gas chromatography

The objective of this study was to develop a method for the quantitative analysis of the methylamine concentration in fermentation broths of Hyphomicrobium zavarzinii ZV 580 cultures. For this purpose an established method for the quantification of free amino acids in such matrices was adapted and validated. The detection limit was 10 microM, the calibration curve showed good linearity (R2=0.9998) in the concentration range between 0.1 and 8 mM. The standard deviation of the injection-to-injection reproducibility (n=10) of the retention coefficient was <1%, that of the peak area<5%. In case of the sample-to-sample reproducibility (n=8), the standard deviation was <5% for the retention coefficient and <10% for the peak area. The validated method was successfully applied for monitoring a fed-batch bioprocess (starting volume: 8L, initial methylamine hydrochloride concentration: 10 mM) producing a dye-linked formaldehyde dehydrogenase in H. zavarzinii ZV 580.     

Improve method for isolation of rat liver plasma membrane

An improved method for the isolation of plasma membrane from rat liver is presented.Gentle homogenization of perfused livers in buffered isotonic KCI, followed by direct flotation of a low-speed nuclear pellet through a discontinuous sucrose density gradient results in a 32% yield, and 25-fold enrichment for the plasma membrane marker, phosphodiesterase I, in a crude plasma membrane fraction. This fraction contains less than 1% of the mitochondria, and endoplasmic reticulum present in the original homogenate, but is more heavily contaminated with lysosomes and Golgi membrane.Vigorous mechanical disruption of this material, followed by a second discontinuous sucrose density gradient, gives a light plasma membrane fraction with an 80-fold purification and 20% yield of phosphodiesterase I over the original homogete (with further reduction of contaminants).     

Simple liquid-chromatographic method for Nile Red quantification in cell culture in spite of photobleaching

Nile Red fluorescent marker is widely-used for different purposes, such as staining cell structures and for the visualization and localization of colloidal drug carriers. However, when fluorescence-dependent imaging or quantification is performed, the risk of inexact results is increased due to photobleaching. The proposed, simple quantification method of using an HPLC-UV-Vis system allows the determination of Nile Red even in photobleached samples. The intra- and inter-assay accuracies for all analytes were found to be within 94.9 and 100.8%, respectively, of target values. When samples underwent photobleaching by laser, UV-Vis detection varied at around 99+/-5%, whereas fluorescence decreased down to 86%. Such results show this method to be interesting for approaches where quantification should be performed after analysis such as fluorescent imaging.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

ABP1: An auxin receptor for fast responses at the plasma membrane

Auxin-binding protein 1 (ABP1) is an auxin receptor for responses not primarily regulated by gene regulation. One fast response is protoplast swelling. By using immunological ABP1 tools we showed that the highly conserved box a is not alone important for auxin binding. Box c is another part of the auxin binding domain.¹ Here we present a novel method to analyze auxin-induced, ABP1-mediated effects at the plasma membrane on single cell level in vivo. The fluorescence of FM4-64 in the plasma membrane is reduced by auxin and this response is mediated by ABP1. This method indicates a functional role of ABP1 at the plasma membrane.Key words: Auxin-binding protein 1, auxin, receptor, protoplast, plasma membrane, FM4-64     

Flow cytometry is a new method for the characterization of intestinal plasma membrane.

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.     

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambda(ex)=330 nm, lambda(em)=460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were 相似文献   

A rapid gas chromatography-mass spectrometry method for quantification of alkylresorcinols in human plasma

Alkylresorcinols (ARs) are phenolic lipids that among foods are found almost exclusively in whole grain and bran products based on wheat and rye. They have been suggested to be used as selective biomarkers of whole grain wheat and rye intake and, thus, may serve as an alternative/complement to commonly used dietary assessment methods in epidemiological studies. For such investigations where analysis of large numbers of valuable samples is required, rapid, sensitive, and repeatable methods are essential. In this article, we describe a rapid and sensitive gas chromatography-mass spectrometry (GC-MS) method for quantification of AR homologues C17:0, C19:0, C21:0, C23:0, and C25:0 in human plasma. The method uses Oasis MAX solid phase extraction cartridges for sample cleanup. A plasma sample of 0.2 ml could be used without preincubation with water. Samples in the range of 7 to 8750 nmol total AR/L were successfully analyzed with the method described, and the average total AR recovery within the reported range was 92 ± 12%. The within- and between-day precision values of total AR concentration in a quality control sample, determined as the coefficients of variation, were on average 7 and 10%, respectively. Approximately 30 to 50 samples could be analyzed within 1 day. The improved GC-MS method presented can be used for rapid analysis of ARs in relatively small sample volumes.