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Biomedical subjects

R Rigler

Publications and source records attributed to R Rigler.

At least 37 records · Page 2Linked to original sources
PubMed2000

Hydrodynamic flow profiling in microchannel structures by single molecule fluorescence correlation spectroscopy

In this paper we demonstrate high spatial resolution hydrodynamic flow profiling in silicon wafer based microchannels using single molecule fluorescence correlation spectroscopy (FCS). We have used confocal fluorescence microscopy to detect single tetramethylrhodamine (TMR-4-dUTP) biomolecules traversing a approximately 1 fL volume element defined by an argon laser beam focus. By elevating a (approximately 10(-10) M) reservoir of diluted analyte, a continuous hydrodynamic flow through the microstructure could be accomplished. The microchannel was then scanned with a diffraction-limited focus in approximately 1-microm steps in both the vertical and the horizontal directions to determine the flow profile across a 50 x 50 microm2 channel. The flow profile measured was parabolic in both dimensions, thereby showing a Poiseuille laminar flow profile. Future microstructures can hereby be nondestructively investigated with the use of high spatial resolution confocal correlation microscopy.

Journal Article↗
PubMed2000

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy.

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.

Liposomes↗
PubMed2000

Evidence for specific interactions between kavain and human cortical neurons monitored by fluorescence correlation spectroscopy.

Specific interactions between a tetramethylrhodamine-labeled kavain derivative and human cortical neurons were found for the first time using fluorescence correlation spectroscopy (FCS). Human cortical neurons, cultivated in Nunc chambers, were incubated with 1 nM of the dye-labeled kavain derivative. A total binding of 0.55 nM was found after an incubation period of 60 min. 50% of the total binding was specifically displaced in the presence of 1 microM non-labeled (+)-kavain. Evidence for these specific interactions was verified by a saturation experiment. Both the non-linear Scatchard plot and the n value of 1.58 +/- 0.07 in the sigmoid Hill plot indicate binding sites with different binding affinities.

Cells, Cultured↗
PubMed2000

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.

Apoptosis↗
PubMed2000

Role of C-peptide in human physiology.

The C-peptide of proinsulin is important for the biosynthesis of insulin but has for a long time been considered to be biologically inert. Data now indicate that C-peptide in the nanomolar concentration range binds specifically to cell surfaces, probably to a G protein-coupled surface receptor, with subsequent activation of Ca(2+)-dependent intracellular signaling pathways. The association rate constant, K(ass), for C-peptide binding to endothelial cells, renal tubular cells, and fibroblasts is approximately 3. 10(9) M(-1). The binding is stereospecific, and no cross-reaction is seen with insulin, proinsulin, insulin growth factors I and II, or neuropeptide Y. C-peptide stimulates Na(+)-K(+)-ATPase and endothelial nitric oxide synthase activities. Data also indicate that C-peptide administration is accompanied by augmented blood flow in skeletal muscle and skin, diminished glomerular hyperfiltration, reduced urinary albumin excretion, and improved nerve function, all in patients with type 1 diabetes who lack C-peptide, but not in healthy subjects. The possibility exists that C-peptide replacement, together with insulin administration, may prevent the development or retard the progression of long-term complications in type 1 diabetes.

Amino Acid Sequence↗
PubMed2000

Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy.

We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine B-labeled substrate by ribonuclease T1. A gapped heteroduplex substrate - a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides - was immobilized via biotin to a streptavidin-coated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).

Binding Sites↗
PubMed1999

Specific binding of proinsulin C-peptide to human cell membranes.

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with K(ass) > 3.3 x 10(9) M(-1). Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 x 10(-4) s(-1). The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative D-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Binding, Competitive↗
PubMed1999

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.

Algorithms↗
PubMed1999

Resolution of fluorescence correlation measurements.

The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.

Animals↗
PubMed1999

Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy.

BACKGROUND: The accumulation of fibrillar deposits of amyloid beta-peptide (Abeta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of Abeta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. RESULTS: The polymerization of Abeta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 microM and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when Abeta was incubated in the presence of Abeta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. CONCLUSIONS: The polymerization of Abeta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by Abeta ligands.

Alzheimer Disease↗
PubMed1998

Fluorescence correlation spectroscopy of enzymatic DNA polymerization.

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

Base Composition↗
PubMed1998

Fluorescence cross-correlation: a new concept for polymerase chain reaction.

In this article we present a new concept for the detection of any specifically amplified target DNA sequences in multiple polymerase chain reactions (PCR) based on fluorescence correlation spectroscopy (FCS). The accumulation of double-stranded target DNA is monitored by the cross-correlated fluorescence signals provided by two amplification primers which are 5'-tagged with two different kinds of fluorophores (Rhodamine-Green and Cy5). Only the amplified target DNA sequence carrying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are required for determining the size and the amplified copy number of specific DNA. The PCR amplification process is started with all ingredients in a single compartment (e.g. of a microtiter plate), in which amplification and measurement are performed. This eliminates the need for post-PCR purification steps. The homogeneous one-tube approach does not depend on fluorescence energy transfer between the fluorogenic dyes. Thus, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifying DNA. The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use. For this purpose, an instrument optimized for two-color excitation and detection of two-color emission has been developed, incorporating the principle of confocal arrangement.

Base Sequence↗
PubMed1998

Single-molecule analysis of restriction DNA fragments using fluorescence correlation spectroscopy.

The cleavage of fluorescence-labeled M13DNA (7250 bp) using HaeIII, HgaI, BsmAI, and BspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 x 10(-15) liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived a practical equation for estimating the number of molecules in the FCS measurements. Under standard enzymatic conditions, HaeIII and BsmAI digested fluorescence-labeled DNA to completion in the range of 8 h, whereas HgaI and BspMI digested the DNA after 40 h. The comparison of recognition sequences suggested that some tagged nucleotides could be inserted between the recognition site and the cleavage site of the slow enzyme group. The decrease in amplitude in the fluorescence correlation function quantitatively monitors the hydrolysis of DNA during the digestion process.

Base Sequence↗
PubMed1998

Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle.

Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these contractions is not determined by the rate of Ca2+ removal or due to an increased Ca2+ sensitivity, but instead is limited by other processes such as deactivation by dephosphorylation or detachment of tension-bearing cross-bridges, possibly regulated by thin filament systems.

Animals↗
PubMed1998

Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces.

Taking advantage of the present day possibilities for ultrasensitive detection by fluorescence, fluorescence correlation spectroscopy (FCS) has over the last ten years emerged as a potentially very powerful technique. In this article we present some results to illustrate the use of FCS for monitoring chemical kinetics on a molecular level and show how, for a wide range of chemical processes, the theoretical treatment can be strongly simplified. The experimental examples given include measurements of ion concentrations and buffer properties, electron transfer reactions, ligand-receptor interactions and diffusion of ligand-receptor complexes in cell membranes. For each of these examples the properties of the FCS technique is discussed in relation to other established techniques used for that particular application. From these examples it is found that FCS can offer important complementary information and, due to the extreme sensitivity of the technique, new information not yet explored by other methods.

Buffers↗
PubMed1997

Conformational fluctuations in single DNA molecules.

Measurement of fluorescent lifetimes of dye-tagged DNA molecules reveal the existence of different conformations. Conformational fluctuations observed by fluorescence correlation spectroscopy give rise to a relaxation behavior that is described by "stretched" exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational fluctuations. Fluorescence fluctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational fluctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.

DNA↗
PubMed1997

Confocal spectroscopy in microstructures.

Confocal instrumentation makes it possible to carry out spectroscopic measurements with a very high signal-to-background ratio. The transit of a single fluorescent molecule through the focal point of the light can be monitored with this method. The particle transport can be observed in transparent microchannels. Examples of fluorescence correlation spectroscopy and single molecule handling in microstructures are discussed.

Journal Article↗