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Biomedical subjects

R Uhl

Publications and source records attributed to R Uhl.

At least 19 recordsLinked to original sources
PubMed2006

Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl cyanoacrylate) nanoparticles: revisiting the role of surfactants.

Poly(butyl cyanoacrylate) nanoparticles coated with poloxamer 188 (Pluronic) F68) and also, as shown previously, polysorbate 80 (Tween 80) considerably enhance the anti-tumour effect of doxorubicin against an intracranial glioblastoma in rats. The investigation of plasma protein adsorption on the surface of the drug-loaded nanoparticles by two-dimensional electrophoresis (2-D PAGE) revealed that both surfactants, besides other plasma components, induced a considerable adsorption of apolipoprotein A-I (ApoA-I). It is hypothesized that delivery of doxorubicin to the brain by means of nanoparticles may be augmented by the interaction of apolipoprotein A-I that is anchored on the surface of the nanoparticles with the scavenger receptor class B type I (SR-BI) located at the blood-brain barrier. This is the first study that shows a correlation between the adsorption of apolipoprotein A-I on the nanoparticle surface and the delivery of the drug across the blood-brain barrier.

Adsorption↗
PubMed2002

A ratiometric imaging method for mapping ion flux densities.

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.

Animals↗
PubMed2001

Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates.

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

Amino Acid Sequence↗
PubMed2000

Laser atomic absorption spectrometry of excited Hg in a discharge applying sum frequency mixing of two diode lasers (preliminary results).

Wavelength modulation diode laser atomic absorption spectrometry is applied to the detection of atomic mercury. Transitions from metastable energy levels highly populated in a radio-frequency discharge are induced with laser diodes by use of nonlinear techniques. The wavelength of one strong transition at 365.119 nm with a high oscillator strength is obtained by sum frequency generation of two diode lasers. The cold vapor technique is used to transfer ionic into atomic mercury. The mercury in the vapor phase is transported by an argon stream into the discharge tube. From the time-dependent absorption signals detection limits of 100 ng/L are achieved at this state of research.

Journal Article↗
PubMed1999

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.

Absorption↗
PubMed1997

Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae).

Animals↗
PubMed1997

How Chlamydomonas keeps track of the light once it has reached the right phototactic orientation.

By using a real-time assay that allows measurement of the phototactic orientation of the unicellular alga Chlamydomonas with millisecond time resolution, it can be shown that single photons not only induce transient direction changes but that fluence rates as low as 1 photon cell(-1) s(-1) can already lead to a persistent orientation. Orientation is a binary variable, i.e., in a partially oriented population some organisms are fully oriented while the rest are still at random. Action spectra reveal that the response to a pulsed stimulus follows the Dartnall-nomogram for a rhodopsin while the response to a persistent stimulus falls off more rapidly toward the red end of the spectrum. Thus light of 540 nm, for which chlamy-rhodopsin is equally sensitive as for 440-nm light, induces no measurable persistent orientation while 440-nm light does. A model is presented which explains not only this behavior, but also how Chlamydomonas can track the light direction and switches between a positive and negative phototaxis. According to the model the ability to detect the direction of light, to make the right turn and to stay oriented, is a direct consequence of the helical path of the organism, the orientation of its eyespot relative to the helix-axis, and the special shielding properties of eyespot and cell body. The model places particular emphasis on the fact that prolonged swimming into the correct direction not only requires making a correct turn initially, but also avoiding further turns once the right direction has been reached.

Animals↗
PubMed1997

A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas.

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

Animals↗
PubMed1996

Instrumentation for multiwavelengths excitation imaging.

We have developed a fluorescence ratio-imaging system which is based on a 12 bit, 2 MHz slow scan CCD camera and a patented (patent number P 42 28 366.3-52) polychromatic illumination system. The latter produces monochromatic light (12 nm bandwidth) of high intensity (> 3 mW between 300 and 500 nm) and allows one to switch to any wavelength between 260 and 680 nm in less than 3.5 ms in a computer-controlled fashion. The possibility to execute complex wavelength protocols facilitates multiple dye measurements with optimal exposure time for a given wavelength and the return to a dark phase in between exposures. Moreover, it allows sweeping over extended spectral regions in order to determine optimal experimental conditions for a given task. Wavelength selection is performed by a diffraction grating which is mounted onto a galvanometric scanner. The grating is illuminated by white light from a 75 W xenon lamp, using exclusively reflective optics, and the diffracted monochromatic light is coupled into the microscope by means of a single fibre quartz light guide. The epifluorescence optics, a special, achromatic, aplanatic UV condensor, image the exit face plate of the fibre into the specimen plane of an inverted microscope. This 'critical illumination' yields better homogeneity in the specimen plane than the classical Köhler illumination. Thus, with the Zeiss Fluar objective 40 x, NA = 1.3, fluence rates close to 10(23) photons m2 s-1 may be achieved at 340 nm. A DOS programme has been written in 'C' which controls both the monochromator and slow scan imaging system. It can acquire up to 13 full frames per s, and in its binning and skipping mode up to 100 subframes per s may be acquired. The frame-transfer structure of the chip allows one to acquire images at wavelength 'B' while simultaneously writing image data previously acquired at wavelength 'A' into the computer.

Animals↗
PubMed1994

On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii.

This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins.

Animals↗
PubMed1993

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.

Animals↗
PubMed1992

Pullout strength comparison of two methods of orienting screw insertion in the lateral masses of the bovine cervical spine.

We undertook a biomechanical study to compare the pullout strength of 3.5-mm AO screws placed in two different orientations within the bovine cervical spine. The first set of screws were oriented obliquely and passed through the lateral mass, as recommended by the AO group. The orientation of the second set was anterior to posterior through the lateral mass, as recommended by Roy-Camille. All screw holes were drilled and tapped by a spinal surgeon experienced with both techniques. Pullout force was measured on an Instron materials testing machine using a self-centering screw-holding chuck and loading rate of 0.833 mm/sec. Although the bone strength in the Roy-Camille orientation was greater (46.7 N/mm versus 36.1 N/mm, p < 0.05), the overall mean pullout force for the AO orientation was greater (607 N versus 471 N, p < 0.025) due to the longer length of bone available for screw purchase (17.0 mm versus 10.3 mm).

Animals↗
PubMed1990

Watching G proteins at work.

It has been known for over a century that rod photoreceptors in the living retina contract and swell in response to light. Although it is still not known whether this structural light-response is of any functional significance, it has recently been possible to correlate the underlying molecular processes with the activation and deactivation of the photoreceptor G protein, transducin. The technique of light-scattering allows the monitoring of minute changes in cell dimensions, and using this non-invasive experimental approach it can be shown that certain properties of the coupling between transducin and rhodopsin are different in a structurally well-preserved system as compared with rod material used for conventional biochemical studies. Thus, not unlike a psychiatrist, who often learns more about a patient's 'interiors' by observing the body language than by direct interrogation, a biochemist, studying the 'body language' of a cell, may extract information about delicate 'cell interior processes' that would be perturbed by more direct experimental approaches.

Animals↗
PubMed1989

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction.

The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling.

Animals↗
PubMed1989

In vitro dark adaptation and preservation of electrical light responses in the retina from bovine eyes.

A method is described which allows the in vitro dark adaptation of rod photoreceptors from cattle eyes, enucleated under ambient light in the slaughterhouse. Without in vitro dark adaptation these eyes are light adapted and cannot be used for certain delicate biochemical studies and for an electrophysiological characterisation of rod responses. The method is very simple and yields large amounts of dark adapted retinal material, allowing experiments that require bulk amounts of photoreceptor cells. The only source of dark adapted photoreceptors so far have been retinae from dark adapted laboratory animals, which had to be killed and processed under infrared light. Eye cups were opened under red light as soon as possible after their enucleation. Their vitreous humor was removed and their retina thoroughly rinsed with ringer's. Then the eye cup was placed in a moist, light-tight box, where dark adaptation took place. Photoreceptors could thus be kept alive for more than 24 h without showing signs of deterioration. Humidity and free access of oxygen to the retina were the only prerequisites for their survival. The physiological intactness of the photoreceptors and their degree of dark adaptation was demonstrated by measuring mass receptor potentials (ERGs). A simple device is described which can be used for the electrophysiological characterisation of these eyes.

Adaptation, Physiological↗
PubMed1989

Optical probes of intradiskal processes in rod photoreceptors. I: Light-scattering study of ATP-dependent dark reactions.

ATP can cause dramatic structural changes in the outer segment of rod photoreceptors. These changes can be visualized by means of a concomitant light-scattering signal AD, a decrease in scattered light intensity of over 20%. The large size of the signal suggests that major structural changes occur. The underlying molecular events may reflect an important, yet still unknown, part of the photoreceptor machinery. AD signals reflect ATPase-driven transmembrane events which occur in and at the disk membrane. Their only structural prerequisite is the structural integrity of the disk compartment. The angular dependence of AD, which can be mimicked by an osmotically-induced disk-swelling, suggests that the disk compartment swells during the production of the AD signal. AD signals proceed with first-order kinetics (half-life = 1 min at 20 degrees C and ATP concentrations of greater than 100 microM) and are accompanied by the hydrolysis of approximately 4 mol ATP (mol rhodopsin)-1. The AD signal is inhibited by a number of transport ATPase inhibitors (quercetin, NBD.Cl, vanadate, DCCD), but not by oligomycin, azide and ouabain. The sensitivity to DCCD, together with the fact that except magnesium no other cation has to be present, points to a proton translocation. This proton transport appears to be electrogenic, since AD signals require the presence of a permeant anion. In physiological saline this is chloride, and the chloride flux is facilitated by a DIDS-sensitive anion transport unit in the disk membrane.

Adenosine Triphosphatases↗