Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.     

DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay

Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu²⁺ and Pb²⁺ ions. The detection sensitivity can be significantly improved to 1 nM by using a “nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response.     

Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.     

A new determination of the shear modulus of the human erythrocyte membrane using optical tweezers.

Optical tweezers are used to apply calibrated forces to human erythrocytes, via small silica beads bound to their membrane. The shear modulus mu of the membrane is inferred from measurements of the cell deformation in the small strain linear regime. We find the same result mu = 2.5 +/- 0.4 microN/m for both discotic and nearly spherical swollen cells. This value is smaller than the one deduced from micropipettes experiments. However the two methods do not operate in the same deformation regime and are not expected to lead to the same result.     

A versatile assay for the accurate,time-resolved determination of cellular viability

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.     

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

A colorimetric assay for determination of cell viability in algal cultures

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.     

High-performance liquid chromatographic assay with fluorescence detection for the determination of cephaeline and emetine in human plasma and urine

A high-performance liquid chromatographic assay method for the quantitation of ipecac alkaloids (cephaeline and emetine) in human plasma and urine is described. Human plasma or urine was extracted with diethylether under alkaline conditions following the addition of an internal standard. Concentrations of alkaloids and internal standard were determined by octadecylsilica chromatographic separation (Symmetry C₁₈ columns, plasma analysis; 15 cm×4.6 mm I.D., 5 μm particle size, urine analysis; 7.5 cm×4.6 mm I.D., 5 μm particle size). The mobile phase consisted of buffer (20 mmol/l 1-heptanesulfonic acid sodium salt, adjusted to pH 4.0 with acetic acid)–methanol (51:49, v/v). Eluate fluorescence was monitored at 285/316 nm. The lowest quantitation limits of cephaeline and emetine were 1 and 2.5 ng/ml, respectively, in plasma, and 5 ng/ml in urine. Intra- and inter-day relative standard deviations were below 15%. The assay is sensitive, specific and applicable to pharmacokinetic studies in humans.     

Clinical assay of the human erythrocyte lactate transporter. II. Analysis and display of normal human data

We have applied a lactate efflux assay to human red cells at two temperatures and with initial lactic acid loads up to 8 mM, metabolically generated. Efflux was about 1.5 times faster at external pH of 8.5 than at 7.5; the latter was the standard pH used thereafter. Multiple lactate loads in a single blood specimen demonstrated clear evidence of saturation kinetics at both pH levels, since the efflux rate did not increase proportionally with the lactate load. Best-fitting rectangular hyperboles were determined for 129-131 assays from 43 volunteers at 20 degrees and 30 degrees. In most cases high and low lactate loads permitted a two-point evaluation of saturation kinetics, and a positive indication was obtained in 88 of 89 tests. The apparent efflux Km and Vm values may be influenced by pH as well as by lactate levels and cannot be taken as rigorous, although they agree reasonably well with literature data on influx and exchange velocities. The data displayed a Hill constant of 1, a 30 degrees/20 degrees velocity ratio of 2.7, and no significant clustering by sex or age. A single assay with initial lactate level above 5 mM at 30 degrees should be sufficient to identify cases with a defective transporter, using the 95% tolerance limits developed in this report.     

Two-dimensional fluorescence spectroscopy – a new tool for the determination of plant cell viability

 Two-dimensional fluorescence spectroscopy (2D-FS) has been used as a new method for determining the viability of tobacco cells (Nicotiana tabacum L.). Both horizontal beam geometry and a vertical set-up achieved with bifurcated fibres were tested. The latter arrangement enabled us to avoid the negative effect of cell sedimentation. Incubation of a tobacco BY-2 cell suspension with dimethylsulfoxide (DMSO) (0–10% v/v) resulted in cell samples differing in their viability – from fully viable (0–2% DMSO) to totally non-viable (8–10%DMSO). The validity of determining viability by means of measuring cell esterase activity by 2D-FS using fluorescein diacetate as a fluorogenic substrate was verified by comparison with microscopic evaluation of fluorescein fluorescence as well as with the routinely adopted trypan blue exclusion test. Received: 6 June 2000 / Revision received: 9 October 2000 / Accepted: 9 October 2000     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

A standardized assay is described for the simultaneous determination of Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and Ca²⁺-ATPase in human erythrocyte (RBC) membrane preparations. Membranes were prepared by lysis of RBCs in hypotonic buffer, and ATPase activity assays were based on the measurement of ³²P-labeled inorganic phosphate release from [γ-³²P]ATP. The results obtained by this method were compared with those of colorimetric determination of inorganic phosphate and of ATP hydrolysis with high-performance liquid chromatography. The activity of the three enzymes was measured in RBC membranes obtained from 30 normal subjects. Repeated sampling of individuals over a 4-month period showed that interindividual differences were substantial, but that in each individual enzymatic activity was maintained in a narrow range by presumed homeostatic mechanisms. Statistical analysis of the data showed no interdependence of the three enzymes; a correlation of activity with age, sex, or phase of the menstrual cycle was not apparent. The values obtained for the Ca²⁺-ATPase did not follow a normal distribution, and it is suggested that this enzyme has two phenotypic variants. The described method is sufficiently precise and economical to be recommended for adoption as standard procedure in clinical research.     

A fluorescence correlation spectroscopy-based enzyme assay for human Dicer

Here, we present an in vitro assay based on fluorescence correlation spectroscopy (FCS), which allows investigation of the kinetic behaviour of human Dicer. The assay is based on the different mobilities of substrate and product. The change of substrate mobility was independent of the choice of the fluorescence label, allowing exclusion of non-specific photophysical artefacts. Dicer and RNase III cleavage led to different product diffusion times. Single-stranded RNA did not change its mobility after cleavage by both double-strand-specific RNases. In agreement with the literature, the RNase activity of Dicer could be inhibited by substituting Ca2? for Mg2?. In a defined system of two diffusion species of similar label and mobility differences, such as substrate and product, the linearity of the assay could be proven. An FCS-based enzyme assay is proposed, which allows monitoring of Dicer activity with high specificity in vitro.     

Novel liquid chromatographic assay for the low-level determination of apomorphine in plasma

A novel HPLC assay which is rapid, reproducible and sensitive has been developed for the analysis of apomorphine in plasma. The assay incorporates boldine as an internal standard, and uses solid-phase extraction on C₁₈ mini-columns for sample clean-up and concentration, so enabling quantitation of apomorphine at 500 pg/ml using fluorescence detection (λ_ex 270 nm, λ_em). The HPLC assay comprised a 25 cm-long Techopakk C₁₈ column and a mobile phase of (0.25 M sodium dihydrogen phosphate plus 0.25% heptane sulphonic acid, to pH 3.3 with orthophosphoric acid) containing 30% (v/v) methanol and 0.003% (w/v) EDTA, run at a flow-rate of 1.5 ml/min. Calibration plots prepared in plasma were linear over the range 1–30 ng/ml, (limit of quantitation (LOQ)=490 PG/ML) with R.S.D. of 0.05% and R.E. of 5.0% at the level of 1 ng/ml. Preliminary pharmacokinetic data from two patients given apomorphine by 12 h subcutaneous infusion (patient A dose=35 mg and patient B dose=141 mg) showed apomorphine elimination from plasma to fit a two-compartment model, with initial half-lives of 8.2 and 46.6 min, elimination half-lives of 76.4 and 166.5 min and area under the plasma concentration-time curve (AUC) values of 236 and 405 ng h/ml, respectively.     

Clinical assay of the human erythrocyte lactate transporter. I. Principles, procedure, and validation

A clinically applicable method for the assay of lactate efflux from human red cells has been developed and described in detail. It requires only small volumes of blood and routine chemicals, and evaluates the process under physiological conditions and direction of lactate loading and transport. The decline of red cell lactate level fit a first order decay curve reasonably well, and better than the fit to zero order or second order plots. Bias is controlled by the use of least-squares curve fitting for all assays, and constraints on the elimination of outlier points. The assay shows a variety of inhibitor effects that may be considered typical for this transporter: potent inhibition by p-hydroxymercuribenzoate, but not by other types of sulfhydryl reagents; marked inhibition by phloretin, quercitin, and 1-fluoro-2,4-dinitrobenzene; lack of inhibition by the amine-reactive agents that block the chloride/carbonate exchanger, DIDS and SITS; and reversible competitive inhibition by alpha-cyano-4-OH-cinnamic acid. Harmaline and N-I-succinimide also produced effective inhibition. The assay also demonstrated transacceleration of L-lactate efflux in the presence of external additions of D-lactate, glycollate, iodoacetate, fluoropyruvate, and bromopyruvate, which are substituted monocarboxylates like lactate, but not by iodoacetamide or L-alanine. Such activation is a manifestation of a macromolecular carrier in operation, and cannot be explained by a pore or channel. These findings satisfy all reasonable criteria for a satisfactory and sensitive lactate transporter assay, which should be adequate to evaluate volunteers and patients for the normal range of this carrier, and to seek possible deficient states.     

Thermal inactivation studies of normal and variant human erythrocyte carbonic anhydrases by using a sulphonamide-binding assay

Heat-inactivation studies were carried out on the two primary erythrocyte carbonic anhydrase isoenzymes, CA I and CA II, and the secondary isoenzyme of CA I, CA I (+1). In addition, two genetic variants of human isoenzyme CA I, CA Id Michigan (100 Thr-->Lys) and CA If London (102 Glu-->Lys), and one variant of isoenzyme CA II, CA IIh (251 Asn-->Asp), were similarly analysed. The first-order rate constants and Arrhenius plots for these six enzyme forms showed that (1) isoenzyme CA II is more heat-stable than CA I, (2) isoenzyme CA I (+1) is less heat-stable than CA I, (3) the variants CA IIh and CA If London are less heat-stable than the normal enzymes, and (4) isoenzyme CA Id Michigan is more heat-stable than normal CA I. From the values of the slopes of the Arrhenius plots, the energy of activation (E(a)) for each isoenzyme and isoenzyme variant was determined, and the following thermodynamic activation parameters were calculated at 55 degrees C: the free energy of activation (DeltaG(double dagger)), the activation enthalpy (DeltaH(double dagger)) and the activation entropy (DeltaS(double dagger)). The DeltaG(double dagger) for the enzymes shows a relative constancy with compensating variation in DeltaH(double dagger) and DeltaS(double dagger). When the values for DeltaH(double dagger) are plotted against DeltaS(double dagger), an increase in DeltaH(double dagger) involves a concomitant increase in DeltaS(double dagger).     

Continuous fluorescence assay for lecithin:cholesterol acyltransferase using a water-soluble phosphatidylcholine.

A water-soluble fluorescent phosphatidylcholine, 1,2-bis[4-(1-pyreno-butanoyl]-sn-glycero-3-phosphocholine (DPybPC) has been used to develop a sensitive, continuous assay for pure lecithin:cholesterol acyltransferase (LCAT) in solution. The monomeric substrate allowed us to examine the reaction of LCAT in the absence of a lipid/water interface in terms of the sensitivity of the enzymatic reaction to anions, ionic strength, apolipoproteins A-I and A-II, and a series of lysophosphatidylcholines and fatty acids. In contrast to the reaction of LCAT with aggregated phosphatidylcholines, the reaction of DPybPC with LCAT was not significantly affected by anions, ionic strength, nor apolipoproteins, indicating that these are only effectors of the interfacial reaction. Lysophosphatidylcholines and fatty acids inhibited LCAT in a chain-length-dependent manner below the critical micellar concentrations of these amphiphiles, indicating that the products of the LCAT reaction can bind to the enzyme and affect its kinetics even in the absence of an interface.     

Dissection of the human erythrocyte spectrin molecule using monoclonal antibodies.

One IgM and three IgG monoclonal antibodies specific to band 1 of human erythrocyte spectrin have been characterised. The antigenic sites of the IgG antibodies have been identified and mapped by radioimmune labelling of tryptic fragments of spectrin fractionated by SDS slab gel electrophoresis and blotted onto nitrocellulose filters. The binding site of one of these antibodies has also been directly visualised in the electron microscope after low-angle shadowing of the antibody-spectrin dimer complex, and lies at that end of the dimer which is responsible for tetramer formation.