Search PubMed⌕ Search

Biomedical subjects

J Greve

Publications and source records attributed to J Greve.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cholesterol, phospholipid, and protein changes in focal opacities in the human eye lens.

PURPOSE: Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). METHODS: Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. RESULTS: Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobicity of tyrosine residues showed a significant decrease, and a change in the tryptophan indole ring angle was found. The changes were abrupt and sharply delineated focal opacities. TEM confirmed this sharp boundary and showed that the opacities were densely packed with vesicles of varying size and electron density embedded in a homogenous matrix. CONCLUSIONS: The Raman and TEM analyses of opacities showed that early cataractogenic events led to disruption of fiber membranes, formation of vesicles from the membrane constituents, and protein changes. The aberrant morphology of the membranes enveloping the focal opacities may have segregated the affected fibers from the surrounding normal tissue, thus explaining the stationary or slowly progressing character of these opacities.

Adult↗
PubMed1997

Experimental and model investigations of bleaching and saturation of fluorescence in flow cytometry.

We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein-isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate kb1 = 6 x 10(3) s(-1), triplet state population rate k12 = 2 x 10(5) s(-1), and depopulation rate k20 = 5 x 10(4) s(-1). The fluorescence kinetics of FITC- and PE-labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water.

Flow Cytometry↗
PubMed1997

Intracellular carotenoid levels measured by Raman microspectroscopy: comparison of lymphocytes from lung cancer patients and healthy individuals.

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells.

Adenocarcinoma↗
PubMed1997

On refraction in Monte-Carlo simulations of light transport through biological tissues.

To obtain reliable results from Monte-Carlo simulations of light scattering experiments, a statistically accurate procedure for positioning the photons after refraction between two different scattering media is necessary. Two statistically equivalent algorithms for calculating the position of the photons immediately after crossing an interface are described and justified.

Algorithms↗
PubMed1997

[Assessment methods for vocational integration of disabled persons--from ERTOMIS methods to the IMBA information system].

The background for the development of actual assessments for evaluation of vocational integration of disabled people (i.e., EAM, IMBA) in Germany is described. Resulting perspectives for future procedural approaches are presented. So far, the EAM, Ertomis Assessment Method, approach has gained limited acceptance only. In light of WHO and European impulses, as well as the economic constraints at hand, it remains to be seen whether the IMBA information system will turn out a practical and problem-focussed tool. A critical review is done to provide constructive suggestions for further applications of IMBA.

Disability Evaluation↗
PubMed1996

The critical sensor: a new type of evanescent wave immunosensor.

A new planar waveguide immunosensor has been developed in which adsorption at a surface, changing the refractive index contrast, is measured. In this "critical" sensor the change in the effective refractive index contrast is transducted to a shift of the critical reflection angle. The sensor's theoretically. In addition, an experimental sensitivity evaluation on the basis of several immunosensing experiments is presented. The obtained lower detection limit is 2 x 10(-2) nm in adlayer growth, equivalent to 12 pg/mm2 of analyte coverage. This sensitivity is comparable to the performance of the surface plasmon resonance sensors or the grating coupler sensors. However, the "critical" sensor has some advantages. These are mainly the ease of fabrication and adjustment prior to a measurement, and the fact that for an experiment no metal layer has to be used.

Antigen-Antibody Reactions↗
PubMed1996

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy.

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Biotin↗
PubMed1996

Aging affects the conformation of cholesterol in the human eye lens.

The distribution of 3-beta-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-beta-hydroxysteroid-specific probe filipin. This non-destructive technique allow a spatially very detailed study of the distribution of 3-beta-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-beta-OH-cholesterol distribution along the optical axis. The 3-beta-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-beta-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-beta-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.

Adolescent↗
PubMed1996

Toothpick perforation of the intestine diagnosed by a small bowel series.

We report the case of a 50-yr-old man who unknowingly swallowed a double-pointed toothpick that ultimately perforated the distal small bowel over a period of several weeks. Appendicitis and ileitis were sequentially considered as diagnosis. A foreign body was diagnosed on a second small bowel series and removed. It has been noted that, because toothpicks are not radiopaque, radiographic studies are not useful in the diagnostic workup. In the case reported, the toothpick was seen on the contrast radiograph of the small bowel and the correct diagnosis ultimately made.

Appendicitis↗
PubMed1995

Occurrence and a possible mechanism of penetration of natural killer cells into K562 target cells during the cytotoxic interaction.

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo) emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism.

Actins↗
PubMed1995

Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: a quantitative study at the single cell level.

This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura-2-AM) and [pH]in (BCECF-AM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell "escapes" immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3-7.6 (approximately 3 min).

Calcium↗
PubMed1995

New approach to assess the cholesterol distribution in the eye lens: confocal Raman microspectroscopy and filipin cytochemistry.

Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytical tool for the study of some aspects of the molecular organization of cells and tissues with high spatial resolution. Filipin, a polyene antibiotic, specifically binds to cholesterol, and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CRM-filipin approach to study the distribution of cholesterol in the human eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm-1, hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely sensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to the intrinsic anisotropy of membranes in the eye lens and therefore of the cholesterol distribution, a line-scan approach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of four human eye lenses was compared with data from the literature. The combined CRM-filipin approach is a highly specific and sensitive method for the study of cholesterol within cells and tissues. The spatial resolution is high and can be adapted to the desired discriminative power. The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.

Cholesterol↗
PubMed1994

Lissajous-like patterns in scatter plots of calibration beads.

Flow cytometric measurements of light scattering of polystyrene calibration beads revealed remarkable Lissajous-like loops in two-parameter scatter plots. The existence of such loops is shown to be in qualitative agreement with Lorenz-Mie scattering theory of homogeneous spheres. The occurrence of these patterns reflects the extreme particle size dependency of perpendicular light scattering of homogeneous spheres. These effects may give an explanation for the frequently observed phenomenon that polystyrene spheres reveal a relatively large CV in the perpendicular light scattering signals, whereas the CV of the forward light scattering signal is small. We conclude that one should be careful to optimize the perpendicular light scattering channel by minimizing the CV, because there is no linear relationship between instrument alignment quality and the CV of the perpendicular light scattering signals of calibration beads.

Calibration↗
PubMed1994

Changes in actin organization during the cytotoxic process.

Changes in organization of F-actin during the cytotoxic process between NK and K562 cells have been observed and studied using confocal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F-actin content and orientation of F-actin towards the target cell have been observed in conjugated NK cells. The increase in F-actin content probably reflects activation of the NK cell for the killing process. An increase in F-actin content in the conjugated K562 cell, occurring simultaneously with the appearance of filamentous actin structures that often originated/ended at the contact place with the NK cell, was also observed. These changes were delayed compared to the increase in F-actin content in the NK cell and were accompanied by increasing cytotoxic activity. This indicates that they were results of the interaction of the K562 cell with the activated NK cell. The possible role of target cell microfilaments in the cytotoxic process is addressed.

Actin Cytoskeleton↗
PubMed1994

Raman microspectroscopic study of low-pH-induced changes in DNA structure of polytene chromosomes.

The effects of low-pH treatments on DNA structure in polytene chromosomes of Chironomus thummi thummi have been studied by Raman microspectroscopy. Measurements were carried out on chromosomes at low pH and on chromosomes reneutralized after a short exposure to low pH. Protonation of adenine residues and subsequent unpairing of adenine (A) and thymine (T) were found to commence already above pH 3.6 and are completed at pH 2.2. Protonation of the cytosine-guanine base pair also starts above pH 3.6. It leads to an intermediate protonated, most likely Hoogsteen-type, guanine-cytosine base pair. Unpairing of G and C residues starts between pH 2.6 and 2.2 and continues below pH 2.2. Brief treatment of chromosomes at a pH < or = 2.2, i.e., at a pH where both AT and GC base pairs are disrupted, was found to lead to irreversible changes in DNA conformation upon return to neutral pH. These were most pronounced after treatment with 45% acetic acid. More than 10% of the A and T residues was found to have adopted a non-B-DNA conformation. Evidence was found for denaturation of the B-form backbone. The amount of protein extracted from the chromosomes was strongly pH-dependent. Treatment at pH 3.6 did not cause noticeable protein extraction, while treatment with 45% acetic acid extracted more than 50% (by weight) of the chromosomal proteins.

Adenine↗
PubMed1994

Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis.

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime.

DNA↗
PubMed1994

Combined elastic and Raman light scattering of human eye lenses.

The distribution of the scattering coefficient (as defined in the appendix) at a wavelength of 647.1 nm along the visual axis of human eye lenses was investigated using a specially designed set-up for spatially resolved measurements of the intensity of the scattered light. For the same lenses, the distribution of the protein content was measured using confocal Raman microspectroscopy. Data collected by both methods were processed in terms of a recently developed theory of short-range, liquid-like order of crystallin proteins that accounts for eye lens transparency. Seven fresh intact human lenses of varying age have been investigated. In addition, elastic and Raman scattering measurements have been performed on fixed lenses. The main results and conclusions are: (1) Fixation significantly affects the light scattering properties of the eye lens. The average level of scattering increases and a change in the distribution of scattered light intensity along the visual axis occurs. Protein content and average distribution were not altered by fixation. (2) There are significant differences between the distribution of the scattering coefficient for lenses of different ages. For young lenses (18 and 20 years) regions with a low protein content (anterior and posterior cortex) show a higher level of elastic light scattering, while for older lenses (42-78 years old) there is no obvious correlation between the scattering level and protein content. (3) Changes in the level of light scattering along the visual axis of the lens cannot be explained by protein concentration effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗