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Biomedical subjects

M Ameloot

Publications and source records attributed to M Ameloot.

At least 19 recordsLinked to original sources
PubMed2006

EDC-mediated DNA attachment to nanocrystalline CVD diamond films.

Chemical vapour deposited (CVD) diamond is a very promising material for biosensor fabrication owing both to its chemical inertness and the ability to make it electrical semiconducting that allows for connection with integrated circuits. For biosensor construction, a biochemical method to immobilize nucleic acids to a diamond surface has been developed. Nanocrystalline diamond is grown using microwave plasma-enhanced chemical vapour deposition (MPECVD). After hydrogenation of the surface, 10-undecenoic acid, an omega-unsaturated fatty acid, is tethered by 254 nm photochemical attachment. This is followed by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)-mediated attachment of amino (NH(2))-modified dsDNA. The functionality of the covalently bound dsDNA molecules is confirmed by fluorescence measurements, PCR and gel electrophoresis during 35 denaturation and rehybridisation steps. The linking method after the fatty acid attachment can easily be applied to other biomolecules like antibodies and enzymes.

Biosensing Techniques↗
PubMed2005

Store-operated Ca2+-channels are sensitive to changes in extracellular pH.

The sensitivity of store-operated Ca(2+)-entry to changes in the extra- and intracellular pH (pH(o) and pH(i), respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca(2+)-stores were depleted either with 1 mM carbachol (CCH) or with 2 microM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca(2+)-entry while external alkalinization augmented both the CCH- and the TG-induced Ca(2+)-influx. Mn(2+)-quenching experiments revealed that the rates of Ca(2+)-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH(o) 8.2 and slowed down at pH(o) 6.8 with respect to the control at pH(o) 7.4. Alteration of pH(o) between 6.8 and 8.2 did not have any significant prompt effect on pH(i) and changes in pH(i) left the CCH-induced Ca(2+)-entry unaffected. These findings demonstrate that physiologically relevant changes in pH(o) affect the store-operated Ca(2+)-entry in SH-SY5Y cells and suggest that endogenous pH(o) shifts may regulate cell activity in situ via modulating the store-operated Ca(2+)-entry.

Calcium Channels↗
PubMed2005

Impedimetric immunosensors based on the conjugated polymer PPV.

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.

Antigens↗
PubMed2003

Characterization of three human oligodendroglial cell lines as a model to study oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression.

Oligodendrocytes, the myelin-forming cells of the central nervous system, are the target of pathogenic immune responses in multiple sclerosis. Primary cultures of human oligodendrocytes have been used to unravel the cellular and molecular mechanisms of immune-mediated injury of oligodendrocytes. However, these studies are hampered by the limited availability of viable human brain tissue. The present study was aimed at comparing the morphological and biochemical characteristics of the human oligodendroglial cell lines HOG, MO3.13 and KG-1C. We have determined oligodendrocyte-associated features of these lines and analyzed the degree to which they can be used as a model of human oligodendrocytes arrested at specific developmental stages. The oligodendroglial cell lines all exhibited markers of immature oligodendrocytes, such as CNPase and GalC, but not the astrocytic marker GFAP. Differentiation could be induced in HOG and MO3.13 cells, as was seen through a decrease in proliferation, an increase in process extension without formation of myelin sheets and up-regulation of genes associated with mature oligodendrocytes such as MBP and MOG. Microarray analysis revealed the expression of MAG, MOBP and OMG genes in HOG cells. The KG-1C cells displayed poor growth characteristics in the recommended conditions. In conclusion, our data show that the oligodendroglial cell lines HOG and MO3.13 can be used as a model of human oligodendrocytes "arrested" in an immature developmental stage. Culturing in appropriate medium can induce further differentiation of these cells. These cell lines can therefore be applied as a model to study immune-mediated injury of oligodendrocytes in relation to disease.

Cell Culture Techniques↗
PubMed2000

Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells.

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (tau2 = 2.4 +/- 0.2 ns) to that of the short component (tau1 = 0.4 +/- 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.

Animals↗
PubMed2000

Fluorescence lifetime microscopy of the sodium indicator sodium-binding benzofuran isophthalate in HeLa cells.

The behavior of the sodium indicator sodium-binding benzofuran isophthalate (SBFI) is investigated in HeLa cells by time-resolved fluorescence microscopy. The fluorescence relaxation of SBFI in HeLa cells can be described by a triexponential for intracellular sodium concentration ([Na(+)](i)) between 0 and 90 mM. Changes in [Na(+)](i) affect neither the fluorescence relaxation times (0.21, 0. 60, and 2.7 ns) nor the average decay time (2.2 ns). The preexponential factor of the shortest decay time is negative. However, the ratio of the fluorescence excitation signal at 340 nm to that at 380 nm increases with [Na(+)](i). To elucidate the behavior of SBFI in cells, experiments are performed on SBFI in buffer at various concentrations of sodium, potassium, and bovine serum albumin (BSA) and at various viscosities. The fluorescence decay is triexponential only in the presence of BSA. The relaxation times are independent of [Na(+)] and [BSA]. The preexponential factor of the shortest decay time is negative from a certain [BSA] on, which depends on [Na(+)]. The data indicate that interactions with intracellular components rather than microviscosity influence the SBFI behavior in cells. A model is suggested in which the fluorescence intensities are mainly determined by the signals from the Na(+) subsetSBFI and SBFI subsetprotein complexes.

Animals↗
PubMed2000

A simple method for obtaining functionally and morphologically intact primary cultures of the medullary thick ascending limb of Henle's loop (MTAL) from rabbit kidneys.

We describe a simple method for obtaining functionally and morphologically intact primary cultures of cells from the medullary thick ascending limb of rabbit kidneys. After digesting dissected fragments of the inner stripe of the outer medulla with collagenase, a suspension of tubule fragments is obtained, the vast majority of which are medullary thick ascending limb (MTAL) segments. These are identified individually by their morphological appearance and large amounts are collected with a micropipette mounted on a micromanipulator. This ensures maximal homogeneity of the starting material. Monolayers of cells grow out of these MTAL segments after seeding them onto collagen-coated, permeable filter supports. During the week following confluence, the cultures exhibit an apical side-positive transepithelial potential difference. Electron microscopic examination shows a monolayer of polarised cells with characteristics of distal tubular cells. The primary cultures express Tamm-Horsfall protein at their apical surface. Additional evidence for their differentiation and polarisation is the net ammonium influx, which occurs at very high rates across the apical membrane and is much slower across the basolateral membrane, as judged by measurements of intracellular pH. Adenosine 3',5'-cyclic monophosphate (cAMP) production is stimulated by arginine-vasopressin, calcitonin or isoproterenol (all 1 micromol/l). Intracellular calcium signalling is observed after stimulation with 1 micromol/l adenosine, adenosine 5'-triphosphate (ATP) and bradykinin. In addition, we compared these characteristics with those of TALH-SVE cell monolayers, an established immortalised cell line of the same origin.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport.

We report, for the epithelial Na+ channel (ENaC) in A6 cells, the modulation by cell pH (pHc) of the transepithelial Na+ current (INa), the current through the individual Na+ channel (i), the open Na+ channel density (No), and the kinetic parameters of the relationship between I(Na) and the apical Na+ concentration. The i and N) were evaluated from the Lorentzian INa noise induced by the apical Na+ channel blocker 6-chloro-3, 5-diaminopyrazine-2-carboxamide. pHc shifts were induced, under strict and volume-controlled experimental conditions, by apical/basolateral NH4Cl pulses or basolateral arrest of the Na+/H+ exchanger (Na+ removal; block by ethylisopropylamiloride) and were measured with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The changes in pHc were positively correlated to changes in INa and the apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying INa was No. Only the saturation value of the INa kinetics was subject to changes in pHc. pHc-dependent changes in No may be caused by influencing Po, the ENaC open probability, or/and the total channel number, NT = No/Po.

Ammonium Chloride↗
PubMed1997

Major histocompatibility complex class I protein conformation altered by transmembrane potential changes.

The nature of charge distributions in membrane-bound macromolecular structures renders them susceptible to interaction with transmembrane potential fields. As a result, conformational changes in such species may be expected to occur when this potential is altered. We have detected reversible conformational change in the major histocompatibility complex (MHC) class I antigen in the plasma membrane of human JY cells, as monitored by flow-cytometric resonance energy-transfer, upon reduction of the transmembrane potential (depolarization). This change increased the intramolecular energy-transfer efficiency between fluorescent donor- and acceptor-labeled monoclonal antibodies directed, respectively, to epitopes on the light (beta 2-microglobulin) and the heavy chains of the MHC class I antigen. Repolarization of the depolarized samples restored the energy-transfer efficiency to the original values measured before depolarization. Depolarization caused similar relative changes in fluorescence resonance energy-transfer efficiency when Fab fragments were used for labeling MHC class I complex, suggesting that the observed phenomenon is not restricted to whole monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1997

Potential misevaluation of the ground-state dissociation constant from fluorimetric titrations: application to the ion indicators SBFI, PBFI, and fura-2.

A test based on time-resolved fluorescence experiments is proposed to assess the interference of an excited-state reaction with the fluorimetric determination of the ground-state dissociation constant Kd of ion [symbol: see text] fluorescent indicator complexes. If an inflection point occurs in the plot of the fluorescence signal vs -log [ion] in the concentration range of the ion where both decay times are invariant, this inflection point can be associated with the correct Kd. In contrast, the inflection point(s) in the concentration range where the decay times vary cannot be attributed to Kd. The test is applied to the fluorescent ion indicators SBFI (for Na+), PBFI (for K+), and Fura-2 (for Ca2+). In all three cases the decay times are invariant in the physiological concentration ranges of the respective ions, indicating that the fluorimetrically determined Kd values are actually the true Kd values.

Benzofurans↗
PubMed1996

Immunosuppressors inhibit voltage-gated potassium channels in human peripheral blood lymphocytes.

The effects of immunosuppressive agents on the potassium current of human peripheral blood lymphocytes have been studied using the whole-cell patch-clamp technique. Cyclosporin A (10 micrograms/ml), rapamycin (10 micrograms/ml) and FK-506 (2.5 micrograms/ml) reduced the peak K+ current by approximately 40, 30 and 40% of the control, respectively, without any change in the reversal potential of the current. The current inhibition was similar at all membrane potentials studied and was accompanied with an increase in the rate of K+ current inactivation. Membrane potential measurements in current-clamp showed a marked depolarization of the membrane (>10 mV) upon the addition of either immunosuppressor to the cells. Our findings revealed that the voltage-dependent potassium current in human peripheral blood lymphocytes is inhibited by Cyclosporin A and other immunosuppressors, resulting in a depolarized membrane potential.

Cyclosporine↗
PubMed1995

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.

Biophysical Phenomena↗
PubMed1986

Simultaneous analysis of multiple fluorescence decay curves by Laplace transforms. Deconvolution with reference or excitation profiles.

The properties and potentials of the noniterative Laplace deconvolution (LAP2) (M. Ameloot and H. Hendrickx, Biophys. J. 44 (1983) 27) are further investigated. It is shown that LAP2 is exact and that no extrapolations have to be calculated or assumed for the data measured in the actual time window if the impulse response function of the investigated system can be described by a sum of exponentials. The formulas for the LAP2 deconvolution against the measured decay of a reference compound instead of the recorded excitation profile are derived. The procedure for the simultaneous analysis of multiple fluorescence decay curves by LAP2 is described in detail. This global analysis allows one to link any decay parameter, is fast and compares favorably with the nonlinear least-squares iterative reconvolution methods. Because of its short computation time the global analysis by LAP2 provides an efficient way to analyze the fluorescence decay surface in terms of decay associated spectra.

Anthracenes↗
PubMed1984

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. A new approximate solution of the rotational diffusion equation.

We discussed the time-dependence of fluorescent emission anisotropy of a cylindrical probe in membrane vesicles. We showed that, if the motion of the probe were described as diffusion in an anisotropic environment, it would be possible to determine not only the second-rank but also the fourth-rank orientational order parameter from the decay of the fluorescence anisotropy. The approximations involved were based on an interpolation of short-time and long-time behavior of the relevant correlation functions. A general expression was derived for the time dependence of the fluorescence anisotropy in closed form, which applies to any particular distribution model. It was shown to be in good agreement with previously reported results for the cone model and the Gaussian model. Finally, the applicability of the theory to time-resolved and differential phase fluorescence depolarization experiments was discussed.

Diffusion↗
PubMed1984

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes.

Various models for the analysis of time-dependent fluorescence anisotropy measurements were evaluated. The discussion was based on the analysis of pulsed experiments with 1,6-diphenyl-1,3,5-hexatriene embedded in small unilamellar vesicles of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine and in dimyristoylphosphatidylcholine/alpha-lactalbumin complexes. It was shown that a recently proposed model (Van der Meer, W., H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx, H. Schröder, 1984, Biophys. J., 46:515-523) described the data better than did the earlier suggested cone model (Kinosita K., Jr., S. Kawato, and A. Ikegami, 1977, Biophys. J., 20:289-305). This permitted the use of the new model for the estimation of the second- and fourth-rank order parameters on nonoriented systems. The results indicated that a fraction of the probes was oriented perpendicularly to the preferred direction of the lipids. An increase of the rotational correlation times of the fluorescent probe and a higher order of its environment were detected after the interaction of alpha-lactalbumin with the dimyristoylphosphatidylcholine vesicles at acidic pH at 24.2 degrees C.

Animals↗