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Biomedical subjects

J Greve

Publications and source records attributed to J Greve.

At least 73 records · Page 4Linked to original sources
PubMed1994

Immunoreactivity of adsorbed anti human chorionic gonadotropin studied with an optical waveguide interferometric sensor.

A study on the immunoreactivity of adsorbed alpha hCG molecules as a function of substrate hydrophobicity and protein coverage is presented. The experiments were performed with a planar waveguide interferometrical immunosensor. The substrate hydrophobicity and the antibody density were found to be of major importance for the immunoreactivity of the adsorbed antibodies. A combination of a hydrophobic (contact angle > or = 80 degrees) substrate and only a fraction (approximately 0.3) of a monolayer of antibodies gives the highest immunoreactivity. However, the immunoreaction affinity constant of the active antibodies is found to be essentially independent of the substrate hydrophobicity. The experimental results indicate that the orientation and not the conformation of the active antibodies on the substrate is dependent on the substrate hydrophobicity.

Antibodies, Monoclonal↗
PubMed1994

Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.

Binding Sites↗
PubMed1994

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy.

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm).

Animals↗
PubMed1994

[Rehabilitation relevant diagnostic procedures].

Presented are four instruments current in rehabilitation diagnostics. They are investigated concerning their usefulness and merit as well as their possibly specific approach, starting out from the World Health Organization's International Classification of Impairments, Disabilities, and Handicaps (ICIDH), which so far has hardly been commented on in the German-language area, with its effort of enabling severity-specific assessment of need and its extensive exposition on the part of the editor of "problem awareness" in relation to the subsequent planes of consideration. The EAM system is focussed on occupationally integrative, job-specific appraisal, attempting to implement standardized performance/adaptation analysis. This is contrasted with the subjective criteria-based, very consistent IRES questionnaire, which however lacks objective severity appraisal. Additionally dealt with is the "RPK" procedure used in evaluating the rehabilitation of persons with chronic mental illness. Overall, it is found that each of the different instruments has its value and merits, although major aspects of rehabilitation and, above all, integration prognosis require differentiated, systemic appraisal and on-going rehabilitation-specific consideration. What is needed is "understanding" the nature of disease consequences at the different planes of experience.

Activities of Daily Living↗
PubMed1993

Local variations in protein structure in the human eye lens: a Raman microspectroscopic study.

Confocal Raman microspectroscopy was used to monitor local and age-related changes in protein conformation in human eye lenses. In clear human lenses of varying age (range 17-80 years) spectra were recorded along the visual axis, using laser light of 660 nm wavelength. The Raman vibrations in the 650-1750 cm-1 spectral region were analyzed. Difference spectra between central core and different positions along the visual axis were calculated after calibration for protein content using the I(1450) cm-1 CH2/CH3 vibration peak. Tryptophan content was quantified using the peak at 760 cm-1 calibrated for protein. Changes in the 'exposed' vs. 'buried' position of tryptophan were analyzed using the peak heights at I(880) and I(760) cm-1. The difference spectra revealed an excess of tryptophan, tyrosine, phenylalanine, beta-sheet conformation and molecules or molecular groups responsible for a 1425 cm-1 peak in the core region in all lenses investigated. The excess peaks disappeared at about 0.6-0.9 mm below the surface. The tryptophan content increased from superficial to deep layers, levelling off between 0.4-0.8 mm below the surface. Upon aging, the tryptophan content increases in the core not in the cortex. No changes in the 'exposed' vs. 'buried' position of tryptophan were observed. Changes in tryptophan and tyrosine probably reflect the maturational shift from cortex to core in the relative content of alpha, beta and gamma crystallines. The age-related increase in tryptophan in the core may reflect the preferential breakdown by endo- and exopeptidases of alpha-crystallins damaged upon aging. The increase in beta-sheet conformation may indicate a post-translational shift in secondary conformation upon aging. These changes in protein conformation are largely completed in a small superficial zone, i.e., in the early life span of the crystallins.

Adolescent↗
PubMed1993

A flow cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process.

This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity.

Clone Cells↗
PubMed1993

A simple optical fiber device for quantitative fluorescence microscopy of single living cells.

A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is 'opened' and 'closed' can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-loaded K562 target cells bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp.

Calcium↗
PubMed1993

Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy.

The presence and subcellular location of carotenoids in human lymphocyte subpopulations (CD4+, CD8+, T-cell receptor-gamma delta+, and CD19+) and natural killer cells (CD16+) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (approximately 10(-3) M) of carotenoids was found in the Gall body. In CD8+ lymphocytes, T-cell-receptor-gamma delta+ lymphocytes and in natural killer cells carotenoids appeared to be concentrated (approximately 10(-4) M) in the Golgi complex. The concentration of carotenoids in CD19+ lymphocytes was found to be below the present detection limit, which is approximately 10(-6) to 10(-5) M. The results provide new possibilities to investigate the mechanism(s) behind the suggested protective role of carotenoids against the development of cancers.

Antigens, CD↗
PubMed1993

Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process.

We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca(2+)-specific dye Fluo-3, while the other cell type is labeled with the pH-sensitive dye SNARF-1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding--and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2-0.3 pH units). TC do not reveal a significant change in pHi.

Aniline Compounds↗
PubMed1993

Detection of in situ hybridization to human chromosomes with the atomic force microscope.

Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1-79 results in clear marking of the telomere region 1p36. The diameter of the probe p1-79 linked reaction product was 75-100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material.

Chromosomes, Human↗
PubMed1993

Signal processing in slit-scan flow cytometry of cell conjugates.

The design and implementation of a real-time signal processing system for slit-scan flow cytometry is described. The system is used to measure the separate scatter and fluorescence peak heights of 2 adherent cells. Preliminary measurements of changes in the membrane potential induced by interactions between natural killer (NK) cells and their target cells are presented.

Barbiturates↗
PubMed1993

Flow cytometric method for simultaneous detection of lymphocyte-K562 conjugates and immunophenotyping of the conjugate forming cells.

A flow cytometric method for the simultaneous quantification and immunophenotyping of conjugates formed by human peripheral blood lymphocytes (PBL) and K562 cells has been developed. The method uses three fluorescent probes. One of the fluorescent probes (F-18) is used for labeling of PBL prior to incubation with K562 cells. After incubation the cells are treated with monoclonal antibodies labeled with phycoerythrin and Red613, respectively. The combination of F-18 fluorescence and light scattering signals enables identification and quantification of the conjugates while the fluorescence of the monoclonal antibodies provides information about the phenotype of the conjugate forming cells. Results obtained using different monoclonal antibodies are presented. The highest conjugate forming capacity has been found in the CD56+CD8+ population while the CD4+CD8- population has shown the lowest capacity to form conjugates. The influence of a washing step on the conjugate formation is discussed. The possibility to use the method in combination with a cytotoxicity assay is indicated.

Antibodies, Monoclonal↗
PubMed1993

White blood cell differentiation using a solid state flow cytometer.

A flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set-up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche photodiode, which was used to detect the weak scattered light in the orthogonal direction. The new flow cytometer set-up is very small, relatively cheap and yields similar results as a standard flow cytometer set-up using a helium-neon laser and photomultipliers.

Cell Differentiation↗
PubMed1993

Cluster analysis of flow cytometric list mode data on a personal computer.

A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce the large number of subclusters. Thus we combine the advantage of the k-means (speed) with that of the nearest neighbor technique (accuracy). In order to achieve a rapid analysis, no complex data transformations such as principal components analysis were used. Results of the cluster analysis on both real and artificial flow cytometric data are presented and discussed. The results show that it is possible to get very good cluster analysis partitions, which compare favorably with manually gated analysis in both time and in reliability, using a personal computer.

Algorithms↗
PubMed1993

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.

Animals↗
PubMed1992

Ageing and changes in protein conformation in the human lens: a Raman microspectroscopic study.

Using confocal Raman microspectroscopy with laser light of 660 nm the secondary and tertiary conformation of crystallins was studied in human lenses of varying age (20-75 years). Differences in cortical and nuclear proteins in individual lenses and among lenses of different age and differences between small equatorial opacities and adjacent clear sites were analysed using a difference spectrum approach. Intensity calibration allows assessment of local variations in protein content. The main findings and conclusions are as follows. (1) Irrespective of the age of the lens nuclear proteins proved to contain more aromatic amino acids, i.e. tryptophan, tyrosine and phenylalanine. This change most probably reflects differences in crystallin composition between nucleus and cortex as described in biochemical studies. (2) Changes in the amide bands indicate a more pronounced beta-sheet conformation of the nuclear proteins. Taking into account available biochemical evidence this most probably reflects a true post-translational change in the secondary conformation of the crystallins. (3) Cortical proteins in 'old' and 'young' lenses are largely identical indicating that ageing is not accompanied by gross alterations in the transcription/translation of the crystallin genes. (4) 'Old' and 'young' nuclear proteins deviate with respect to the amount of aromatic amino acids, being more abundant in 'young' nuclear proteins. (5) Proteins in small early opacities do not exhibit alterations in conformation. (6) A pronounced peak in the difference spectra in the region 1425-1435 cm-1 for nuclear proteins especially when compared with equatorial cortical proteins may be considered as evidence for the advanced photooxidation of tryptophan and/or deamidation of asparagine from superficial to deep regions of the lens.

Adult↗