PubMed Health⌕ Search

Biomedical subjects

P Mühlig

Publications and source records attributed to P Mühlig.

At least 19 recordsLinked to original sources
PubMed2003

Noninvasive in situ observation of the crystallization kinetics of biological macromolecules by confocal laser scanning microscopy.

High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.

Crystallization↗
PubMed2002

Demystifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis.

The processes taking place during routine chromosome preparation are not well understood. In this study, the morphological changes in amniotic fluid cells, blood lymphocytes, and bone marrow cells in the metaphase stage were examined under an inverted microscope during chromosome preparation. The putative processes that occur during chromosome preparation were simulated in suspension, and the cells were treated with different mixtures of hypotonic solution, fixative, methanol, acetic acid, and water. Evaporation of the fixative was performed under normal atmospheric conditions and under vacuum at different levels of humidity. Freeze fracture electron microscopy was used to analyze the effects of fixative on the cell membrane. Confocal microscopic analysis was used to investigate three-dimensionally the effects of hypotonic treatment on the positions of chromosomes in fixed mitotic lymphocytes. Chromosome preparation-induced changes in the lengths of single chromosomes were also investigated. The results show that chromosome spreading involves significant water-induced swelling of mitotic cells during evaporation of the fixative from the slide, which is a prerequisite for chromosomal elongation, the production of metaphase spreads for chromosome analysis, and the appearance of Giemsa banding patterns. Hypotonic treatment is essential for well-spread metaphase chromosomes because it moves the chromosomes from a central to a more peripheral position in the cell, where they can be stretched more effectively during mitotic swelling. Like mitotic cells, isolated single chromosomes also have their own potential to swell and lengthen during chromosome preparation. We hypothesize that chromosome preparation leads to a genome-wide chromosomal region-specific opening of chromatin structures as GTG-light bands and sub-bands. Living cells may possess a similar mechanism, which is used only to open single chromatin structures to facilitate transcription. We propose the concept of chromosomal region-specific protein swelling.

Cells, Cultured↗
PubMed2000

Lipopolysaccharide induces distinct alterations in the microtubule cytoskeleton of monocytes.

Microtubules are obligate functional elements of almost all eukaryotic cells. They are involved in a broad range of essential cellular functions and structural changes of this system may trigger cell death. Recently, we have reported that lipopolysaccharides inhibit in vitro microtubule formation due to exclusion of microtubule-associated proteins. The distinct epitopes of lipopolysaccharides responsible for these effects and the in vivo relevance of these data are unknown. Therefore, this study was conducted to elucidate the effects of lipid A, the biologically active motif of lipopolysaccharides, on microtubule formation in vitro and to prove whether lipopolysaccharides affect the microtubule architecture of cultured human monocytes in vivo. Despite a dose- and pH-dependent inhibition of microtubule formation by lipopolysaccharides, inhibition of microtubule assembly could be mimicked by lipid A. Near-infrared two-photon microscopy revealed that human peripheral blood monocytes accumulate lipopolysaccharides. A vesicular distribution pattern of lipopolysaccharides within the monocytes was observed. Confocal laser scanning microscopy demonstrated alterations in the microtubule architecture of monocytes after incubation with lipopolysaccharides. Lipid A seems to be responsible for the observed crosstalk between lipopolysaccharides and microtubule proteins. Furthermore, our data indicate that lipopoly-saccharides may affect the microtubule architecture in human monocytes after intracellular accumulation directly. Therefore, we conclude, that the microtubule cytoskeleton is an essential intracellular target for sepsis-relevant bacterial components such as lipopolysaccharides.

Animals↗
PubMed1997

The spreading of metaphases is a slow process which leads to a stretching of chromosomes.

In routine chromosome harvesting of blood lymphocytes it is well accepted that metaphase spreads are obtained from fixed mitotic cells which burst on the surface of slides during the dropping procedure. For confirmation and clarification, fixed mitotic cells were dropped onto coverslips and observed under an inverted microscope during the evaporation of the fixative. Fixed mitotic cells in the metaphase stage first stick onto the surface of the coverslip without changing their three-dimensional shape and they do not burst. Thereafter, when evaporation of the fixative occurs, they slowly flatten until they are spread. This slow process leads to a stretching of chromosomes which may be a prerequisite for high resolution banding patterns. Confocal laser scanning microscopic measurements of the length, thickness, and width of chromosomes after (i) short term evaporation of the fixative, (ii) evaporation of the fixative under routine harvesting conditions and (iii) a prolonged evaporation, confirmed the stretching of chromosomes. The humidity, the temperature, and the drying time of the fixative influence the dynamic flow of the remaining fixative on the slide. This dynamic flow leads to an intensive wash of the fixed mitotic cells with increasing concentrations of acetic acid which is primarily responsible for the better quality of the metaphase spread.

Cell Nucleus↗
PubMed1997

The nature of G-bands analyzed by chromosome stretching.

To investigate the nature of G-banding, chromosome stretching was performed on chromosome 6 at the 400-band level of normal human lymphocytes that had been cultivated and harvested using standard techniques. The GTG-banding patterns of five stretched chromosomes 6 were compared microscopically with each other and found to be identical at the 1,400-band level. A high-resolution ideogram at the 1,400-band level was constructed. The banding pattern at this level appeared to be very regular, with all dark bands at the 400-band level splitting into three to six dark subbands. While the dark subbands observed at the 1,400-band level seem to derive solely from the dark bands seen at the 400-band level, light bands visible at the 400-band level do not split into subbands, which is in contrast to the published (ISCN, 1995) ideograms. The splitting process, which was analyzed on the video monitor in more detail, shows that chromosome stretching is due mainly to the appearance of light subbands flanked by dark subbands. To shed more light on this phenomenon, the staining intensity of the dark bands and their subbands was measured while the chromosomes were stretched from the 400- to the 1,400-band level. At first, staining intensity was found to diminish in inverse proportion to the elongation of the chromosome, but then remained relatively unaffected until the dark subbands were gradually split up. After stretching to the 1,400-band level, these dark subbands were followed by newly appearing small light subbands, which were about the same size as the stretched light bands visible at the 400-band level. The results indicate that, in general, the light bands of human chromosomes are the preferentially stretched chromosome regions and that the resolution-dependent characteristic banding pattern of human chromosomes is mainly based on a fixed hierarchy of the stretchability of chromosomes.

Centromere↗
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↗
PubMed1992

Histomorphometry of focal myocardial lesions by means of the automatic image analysis.

There is an urgent necessity to objectify focal myocardial lesions (fml) before structural changes in muscle cells can be seen light microscopically. Therefore the succinodehydrogenase (SDH) reaction was chosen because it reliably demonstrates the decrease in aerobic capacity already in structurally unchanged cells and distinguishes the latter from the SDH-negative structures. A combined planimetric and densitometric procedure was developed to measure the area density of fml and their 2 constituents mentioned as well as to evaluate the SDH activity in fml and its decrease in muscle cells using the automatic image analysing system QUANTIMET. The verification tests show that both the area density and extinction can be reliably determined with acceptable measuring errors unless the area density is too low. The results of interobserver and repeatability tests are satisfying.

Animals↗
PubMed1991

The microtubule system and the reduplication of microtubule organizing centres in Dictyostelium discoideum.

The microtubule system of normal and microtubule-poisoned amoebae of Dictyostelium discoideum has been investigated both by indirect immunofluorescence with antibodies to microtubule proteins and electron microscopy. Nocodazole, like some other microtubule poisons, destroys most of the microtubules in both interphase and dividing cells resulting in an inhibition of nuclear and cell division. The microtubule organizing centres, however, continue to duplicate once or twice. The daughter organizing centres segregate, they seem to be connected with nuclear material, that splits partly, too, forming more or less extended nuclear clefts. This segregation, at least over short distances, takes place without intranuclear microtubules. Duplication of microtubule organizing centres is not strictly correlated with nuclear division and cytokinesis. Microtubule poisons are able to uncouple these events. Different levels of regulation should be responsible for microtubule organizing centre, nuclear, and cell division.

Animals↗
PubMed1989

Progress in the histomorphometry of the microcirculatory system.

Following a short review of the limits set to the procedures applied so far to measure quantitative changes in wall tissue of microvessels, a new measuring method is presented. It detect morphological reactions of the microcirculatory system on the grounds of changes in the numerical density of selectively visualized microvessels and their classification according to the external diameter by means of the automatic microscopic image analysing system QUANTIMET. Influences of structurally based and/or postmortal changes of the lumen wideness on the measurement are excluded by the automatic subtraction of the lumen area.

Animals↗
PubMed1989

Progress in application of the image analysing system QUANTIMET 720 in the histomorphometry of the microcirculatory system.

The application of automated image analysing methods in the field of histomorphometry has been improved in the last decades. In this paper a new method is presented for the investigation of morphological reactions of the microcirculatory system. It is based on the measurement of quantitative changes in microvessel wall tissue by using the image analysing system QUANTIMET 720. In the measurement model, a microvessel intersection figure is considered as an ellipsis. A procedure is presented allowing the estimation of the wall and lumen area of the microvessel section and the calculation of its radius for a totally collapsed state. From this data, further morphometric parameters are derived. Using this method, the analysis of 500 microvessels including classification and parameter derivation takes about 10 min.

Algorithms↗
PubMed1987

Blood viscosity in patients with bone fractures and long term bedrest.

Eighteen patients with bone fractures and without systemic disease were studied for blood rheology. Blood and plasma viscosities, haematocrit, red cell aggregation and filterability as well as plasma colloid oncotic pressure were measured. Results show that patients exhibit a substantial haemorheological deficit on admission. During bedrest this returns to normal, owing to marked 'autohaemodilution'. It is concluded that trauma causes increased viscosity of blood in these patients. This change could predispose to deep vein thrombosis. However, such a risk is reduced by 'autohaemodilution' which is most probably an effect of immobilization.

Bed Rest↗
PubMed1987

Hypoxia-induced regression of adaptive structural microvessel reactions caused by long-term contraction influences in spontaneously hypertensive rats (SHR).

Chronic effects of hypobaric hypoxia (hb.h.) on the microcirculatory system (m.s.) of skeletal muscle were investigated. A new histomorphometric method was applied to detect the adaptive structural reactions of wall cells to long term changes in contraction performance. Seventeen SHR were exposed to hb.h. from 5 to 18 or 17 to 30 weeks of life, respectively. For controls 19 SHR and 25 normotensive Wistar rats were kept at sea level. The findings document long-term actions of vasorelaxation factors in hb.h. and the ability of the latter to antagonize the intensified vasocontraction factors in SHR. Depending on the stage of hypertension, there are different preferential sides for the actions of both these factors within the various districts of the m.s.

Adaptation, Physiological↗
PubMed1985

[Erythrocyte-associated plasma proteins, a further aspect of erythrocyte function?].

The amount of plasma proteins associated to erythrocytes was determined or calculated by using three different techniques. Thus, there are 3.48 X 10(12) g of protein in a single erythrocyte or 94.12 g of protein respectively in the total of all erythrocytes in a healthy man with a body weight of 70 kg. The mass of plasma protein associated to erythrocytes will decrease in case of immunocomplex aggregates or otherwise denatured plasma proteins being fixed to the erythrocyte surface so that from a purely calculating point of view protein amounts on a scale of up to 46% of all plasma proteins may enter the free blood plasma under extreme conditions. In this, an immediately efficient possibility of compensation is seen by the authors in case of an acute consumption of blood proteins.

Antigen-Antibody Complex↗
PubMed1984

Long-term effects of psychoemotional stress and tread-mill exercise on the microcirculatory system of rats.

To answer the question of whether the increased intensity of tonic or of relaxing influences acting on the microcirculatory system predominate in various chronic psychoemotional stress situations and/or after long-lasting and intense tread-mill exercise, the left-ventricular wall of the heart and the skeletal muscle or pancreas of 49 experimental and 33 control animals were investigated, using a new histomorphometric method. Chronically stressed rats showed an increase of wall thickness in microvessels documenting a long-term increase of contractile performance of wall cells for the development and maintenance of an increased contractile tone; however, there were quantitative differences depending on the manner, intensity, and duration of stress. The opposite occurred in rats exercised by a tread-mill over 6 months, 3 h per day, demonstrating the morphological correlative of metabolic long-term autoregulation. An aversion stress applied simultaneously on these animals effectively antagonized the mentioned effects of exercise.

Animals↗
PubMed1984

Long-term effects of ethanol on coronary microvessels of rats.

To study the hitherto unknown long-term effects of ethanol on the contractile activity of wall cells of coronary microvessels (mv.), 21 experimental and 21 control animals were investigated, using a new histomorphometric method. The pair-fed rats of the experimental group were given a diet containing ethanol which replaced 36% of the total calories for 4 weeks. This resulted in an increase of wall thickness of mv. in all segments of the microvasculature and an augmentation of the number of ATP-hydrolyzing small-calibered mv. The increase of wall thickness of muscularized mv. was interpreted as a structural adaptation to a long-term predominance of vasotonic influences, which were apparently likewise responsible for the increase of ATP-hydrolysis in the smallest mv. The possible reasons for this predominance are discussed and the necessity to include myocardial metabolic factors in pathogenic considerations on ethanol-induced coronary affections is indicated. Such mv. reactions as observed may be considered as a potential contributive pathogenic factor in ethanol-induced cardiomyopathies and hypertension.

Adenosine Triphosphate↗
PubMed1982

Long-term vasotonus behavior of the microvasculatory system of heart and skeletal muscle in rats exercised by swimming.

A new histomorphometric method was used to study the hitherto unknown long-term vasotonus behavior of the arterial microvasculatory system. The right and the left ventricular walls of 29 rats exercised by swimming (48 hours in 4 weeks), and 30 control animals, as well as the musculus rectus femoris of 5 experimental and 5 control animals were investigated. The walls of arterial microvessels of the right ventricular wall and of the skeletal muscle of trained rats were significantly thicker indicating a long-term vasotonus increase. In the microvasculature of the lift ventricular wall no differences were found in comparison to control animals. In view of the vasotonus increase in the right ventricular wall and skeletal muscle, one can assume that tonus-decreasing factors of nearly the same intensity and/or duration of efficiency have opposed the tonic influences in the left-ventricular microvessels, thus preventing a thickening of vessel walls.

Adenosine Triphosphatases↗
PubMed1981

[Use of automated image analysis for screening the efficiency of turimycin-producing Streptomyces strains].

The growth of the surface colonies on solid media is studied with the image analyzing system Quantimet 720 M controlled by a PDP 11 computer. For the bacterial strain Streptomyces hygroscopicus JA 6599 a new differential equation describing the kinetic behaviour of the area of the colony is suggested. It is shown that this time dependence of area describes more realistically the experiment than the function resulting from Pirt's model. Two new constants arise from the growing curve, alpha describes the profile of density of the colony and k0 is correlated to the growing constant of the colony. Both parameters can be correlated to the amount of antibiotics produced and, therefore, it is possible to use this information for the selection procedure of the colonies. A uniform criterion for selection was found for each colony of two mutants of the turimycin-producing strain Streptomyces hygroscopicus JA 6599, which reads: (Formula: see text). The quality of the selection criterion of surface colonies with higher productivity is dependent on the mean value of the growing parameters of all colonies under investigation. Limitations of the selection method arise from the need for great uniformity of environmental conditions, growth medium and a homogeneous spore suspension, which have to consist of a large amount of single spores in order to assure a reproducible growing curve of the colonies. For the last condition a special technique is pointed out.

Agar↗