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Biomedical subjects

K O Greulich

Publications and source records attributed to K O Greulich.

At least 37 records · Page 2Linked to original sources
PubMed1995

Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers.

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10-30 micrometers basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 micrometers) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depend mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 micrometers) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11-110 pN for acropetal and of 18-180 pN for basipetal statolith displacements.

Actins↗
PubMed1995

Wavelength dependence of laser-induced DNA damage in lymphocytes observed by single-cell gel electrophoresis.

Human lymphocytes from a cell culture were irradiated with laser pulses of an Xe-Cl excimer laser (308 nm) or a dye laser at wavelengths from 312 to 640 nm. After conversion of photoinduced DNA damage into DNA strand breaks the "comet assay" (single-cell gel electrophoresis) was used to determine the number of photons required to induce detectable DNA damage. Between 308 and 450 nm the number of photons necessary for DNA damage increases about 10,000 times from 2.14 x 10(8) to 2.85 x 10(12) photons. Between 308 and 318 nm the number of photons necessary to induce detectable DNA damage is proportional to e0.45 lambda. Between 340 and 450 nm it is proportional to e0.05 lambda. No detectable damage was induced at 540 and 640 nm after irradiation with 9900 kJ m-2. Two-photon effects are unlikely to play a significant role.

Cells, Cultured↗
PubMed1995

Transcription specific differences visualized by fluorescence in situ hybridization pattern on interphase nuclei of different cell types.

Application of a "formamide free" and thus "material preserving" in situ hybridization technique using the cDNA of the myf3 gene revealed the following results: Human rhabdomyosarcoma cells, characterized by a high expression of myf3 show intensive hybridization signals in their interphase. RNase treatment prior to hybridization considerably reduces the size of this signals. In comparison, isolated nuclei of human lymphocytes in which no need for the expression of this gene exists, show barely hybridization signals. Correspondingly, RNase treatment had no effect on hybridization pattern at all. In conclusion an increased transcription efficiency of a cell type specific gene is accompanied by a higher hybridization accessibility in the corresponding cell nuclei.

Cell Nucleus↗
PubMed1994

Beware of using small statistical samples when assessing the quality of a DNA library.

DNA libraries often contain very large numbers of clones (from 1000 up to 700,000). Since at present it is impossible to analyze all of these clones, usually statistical samples comprising less than 100 clones are tested. The quality of the library is then assessed by linear extrapolation. Occasionally, full coverage of chromosomal regions by DNA probes is inferred from this. However, this may not be accurate since linear extrapolation is misleading and the statistical samples are generally too small to characterize the libraries. A quantitative model of the distribution of the frequencies of the clones in a library is mandatory for any useful assessment of the quality of the library. Otherwise, it is very difficult to draw useful conclusions from moderately sized samples. Examples from everyday life and formulas are given to determine the quality of a library and useful sample sizes.

Chromosome Walking↗
PubMed1994

Laser micromanipulators for biotechnology and genome research.

The use of lasers for complete micromanipulation of metaphase chromosomes, cells and subcellular structures is reviewed. DNA probes from single microdissected chromosome segments can be prepared using Alu or Adaptor PCR. In plant biotechnology, laser microsurgery can be used to prepare non-enzymatically protoplasts from Medicago sativa. Microgravity can be simulated in the alga Chara by lifting intracellular gravity transmitting elements with the optical tweezers.

Base Sequence↗
PubMed1994

UV-B-laser-induced DNA damage in lymphocytes observed by single-cell gel electrophoresis.

Human lymphocytes from a cell culture were irradiated with 12 mJ pulses (524 J m-2) of an XeCl excimer laser (308 nm). DNA strand breaks were analysed with the "comet assay", a single-cell method. The DNA damage of individual cells could be investigated depending on the energy and the irradiated volume. DNA damage could be detected already after a single laser pulse of 12 mJ. Since the irradiation time is short compared with the DNA repair time, DNA damage is solely dependent on the total number of photons. A quantitative evaluation of the number of damaged cells vs. number of photons can be best explained by a detection threshold model. The calculated threshold is 1.25 x 10(8) photons cell-1. From this and the principal sensitivity of the comet assay, one can estimate that the upper limit for DNA damaging efficiency is 400,000 photons.

Cells, Cultured↗
PubMed1994

New cell biological applications of the laser microbeam technique: the microdissection and skinning of muscle fibers and the perforation and fusion of sarcolemma vesicles.

In a novel approach, the laser microbeam technique was used to selectively perforate the sarcolemma of skeletal muscle fibers, to prepare fragments of myofibrillar bundles of very small dimensions, and to induce fusion of sarcolemma vesicles. Using a highly focused UV laser microbeam with an effective beam diameter of down to 0.5 micron, very small (< 3 microns) myofibrillar fragments with an intact sarcomere striation pattern were obtained. When small amounts of Ca2+ were released in the vicinity of such a fragment by laser-photolysis of the photolabile compound Ca(2+)-nitr-7 the bundle shortened due to the development of calcium-activated force. We also show that very small selected areas from myopathic single muscle cells can be dissected with a precision unmatched by other current techniques. The microbeam was also used to remove very small patches of the sarcolemma of murine skeletal muscle fibers so giving diffusional access to the myoplasmic interior and thus resulting in a "skinning" of the fiber. To ensure that such laser-skinned fiber segments were physiologically intact we determined the Ca(2+)-activated force and caffeine-induced Ca(2+)-release from the sarcoplasmic reticulum. The fibers showed normal characteristics for force production, Ca(2+)-release and uptake by the sarcoplasmic reticulum. To test the effects of the laser microbeam on the muscle membrane directly, we prepared sarcolemma vesicles of skeletal muscle fibers. The vesicles could be selectively perforated with single laser pulses to allow entry of fluorescein isothiocyanate (FITC)-dextran as a fluorescent marker. Adjacent vesicles were caused to fuse by a few pulses at low intensity of the laser microbeam.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The laser microbeam trap as an optical tool for living cells.

Pulsed ultraviolet lasers coupled into a microscope can be used for micromanipulation of cells and subcellular structures. In addition, continuous infrared lasers can be used as ultrafine optical tweezers (or synonymously: optical trap). The pulsed UV lasers (for example excimer lasers or nitrogen lasers) can be used as optical scalpels for the preparation of protoplasts from plant root hairs. The precise microdissection of chromosomes with the laser microbeam provides access to chromosome segments where a specific gene is supported to be localized. From such segments, specific DNA libraries can be prepared for the search after such genes or markers in their environment. With the optical trap contact between effector cells of the immune system and their target cells can be established in a very simple and gentle way. The kinetics of the attack of a natural killer on an erythroleukemia cell can be studied from the first seconds after contact. Isolated plant cells as well as cells in a plant embryo tissue can be perforated and DNA or fluorescent molecules can be injected. From the temperature dependence of laser induced membrane lesions one can obtain predictions on laser induced cell fusion, which can be performed at slightly modified irradiation conditions under total microscopic control. Since focusing into the depth of a cell with an accuracy of better than a micrometer is possible, one can work on subcellular structures in the interior of a cell without opening it. For example, in rapeseed protoplasts subcellular structures such as mitochondria or chloroplasts can be perforated or moved out of their original position. Interestingly, such structures find their way back into the original position after the laser is switched off. From their speed one can obtain estimates on intracellular viscoelasticity.

Animals↗
PubMed1993

Electronic effects on the fluorescence of tyrosine in small peptides.

It is shown for a series of tyrosine-derivatives and tyrosine-containing peptides that the amide group in combination with electron-withdrawing substituents quenches the fluorescence of the phenol moiety. The ammonium group has the strongest electron-withdrawing effect and thus the largest influence on the quenching rate. The peptide group itself does not quench the fluorescence. In a series of peptides with an increasing number of alanines the decreasing quenching efficiency of the peptide group due to the greater distance of the ammonium group is demonstrated. In tyrosine-containing di- and tripeptides a linear correlation between the 13C-NMR chemical shift delta of the C alpha atom of various aliphatic amino acids and the fluorescence-quenching constant confirms the hypothesis that electron-withdrawing and -donating groups are modulating the fluorescence-quenching efficiency of the peptide group. In small peptides the fluorescence lifetime of tyrosine is characteristic for the neighboring amino acids. Using model substances the redox properties of a peptide group and the phenol ring were studied electrochemically. The highest occupied molecular orbital of the tyrosine (1.4 V vs saturated calomel electrode [SCE]) and the lowest unoccupied molecular orbital of the peptide group (-3.12 V vs SCE) have appropriate energies for a photoinduced electron transfer reaction. For solute-quenching experiments quencher molecules can be systematically selected.

Amino Acid Sequence↗
PubMed1991

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

Animals↗
PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗
PubMed1989

Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells.

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the 'core' proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.

Cell Nucleus↗
PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗
PubMed1988

Volume changes during enzyme reactions: indications of enzyme pulsation during fumarase catalysis.

Overall activation volumes for multistep reactions are not usually pressure independent. The present investigation gives a quantitative description of this effect under Theory. Simple relations are obtained which can easily be applied to experimental data and which allow more insight into the dynamics of enzyme reactions. This is demonstrated under Experimental Application for the conversion of fumarate to L-malate catalyzed by the enzyme fumarase. The volume profile of this reaction indicates a pulsation of the enzyme molecule during catalysis. The appendix discusses the question whether Eyring's transition-state theory is an appropriate basis for investigations of this kind.

Fumarate Hydratase↗
PubMed1988

The nasal polyps as a tool for basic research in cystic fibrosis.

Total RNA and mRNA were prepared from cystic fibrosis (CF) and control nasal polyps and nasal epithelial cells. Genomic clones from the chromosomal region of the CF locus were screened by northern blots. A representative cDNA library from nasal polyps was cloned in the vector lambda gt10. For the construction of a physical genomic map around the CF locus single gene markers were isolated from metaphase 1:7q2qter chromosomes by laser micro-dissection and subsequent microcloning. A linkage study with the polymorphic markers met-H, met-D, and pJ3.11 was performed in 53 German CF families with at least 2 children. No significant correlation of any haplotype on the CF chromosomes with the clinical severity of the course of the disease could be observed, which provides evidence that cystic fibrosis is genetically homogeneous.

Chromosome Mapping↗
PubMed1987

Transition of chromatin from the "10 nm" lower order structure, to the "30 nm" higher order structure as followed by small angle X-ray scattering.

Chromatin oligomers undergo a conformational change from a "10 nm" lower order structure at low concentration of salt to a "30 nm" higher order structure, with increasing NaCl or MgCl2 concentration. We have extended our previously reported hydrodynamic and light-scattering measurements of the folding of well-defined chicken erythrocyte chromatin fractions to include a study of the low angle X-ray scattering in solution. We show that it is feasible to identify the folding process with gradual compaction of a chain of freely joined filaments or a worm-like chain, within the limits of all the experimental data obtained. As the ionic strength is raised, the filament length of the oligomer, composed of Nz nucleosomes, decreases. At 75 mM-NaCl, the compacted model chains (Nz = 53) form structures that are, on average, cylindrically shaped with mean diameter 30 nm and length 104 nm. Helical symmetry need not be invoked in the modelling of the folding process and may, in particular, be difficult to establish in chicken erythrocyte chromatin, due to the non-uniform length of the DNA linker connecting the nucleosomes. Concerning the shape of the X-ray scattering profiles at various salt concentrations, it is possible in this way to rationalize two-slope cross-sectional plots, which have also been reported by other workers. Though this description represents a satisfactory conceptual presentation of a wealth of experimental data, it by no means represents a definitive solution to an exceedingly difficult problem.

Animals↗
PubMed1987

Laser-induced fusion of mammalian cells and plant protoplasts.

An ultraviolet-laser microbeam was shown to be suitable for inducing fusion of individually selected plant protoplasts or of B-lymphocytes with myeloma cells. The fusion took place in normal culture medium and the fusogenic condition perturbed the cells only for a fraction of a millisecond. Without manipulating the cell culture except for exposing the cells to laser light, fusion products between preselected individual pairs may be produced.

Animals↗