PubMed Health⌕ Search

Biomedical subjects

P Walther

Publications and source records attributed to P Walther.

At least 19 recordsLinked to original sources
PubMed2003

Recent progress in freeze-fracturing of high-pressure frozen samples.

Pancreatic tissue, bacteria and lipid vesicles were high-pressure frozen and freeze-fractured. In addition to the normal holder, a new type of high-pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low-mass copper platelets, as used for propane-jet freezing. Both the standard and the new suspension holder allowed us to make cryo-fractures without visible ice crystal damage. High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a new type diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and blockfaces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. Additional structures can be made visible by controlled sublimation of ice ('etching'), leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life-like state, preventing the artefacts associated with conventional fixation techniques.

Animals↗
PubMed2003

[Intensive care of delirium syndromes].

Delirium is mental dysfunctions occurring as impaired attentional and memory systems with disturbances of consciousness, affectivity, psychomotor activity and sleep patterns. Numerous factors and underlying diseases may be responsible for these non-specific symptoms. Therefore, a thorough evaluation of preadmission history and current clinical status, supplemented by laboratory and extended technical diagnostic procedures, are always required. If delirium occurs in connection with emergency admission to hospital, an organic disease can most regularly be found. Due to its rapid time of onset and minor side-effects, the intravenous injection of 2.0 g gamma-hydroxybutyric acid is preferred for sedation of extremely agitated patients. Neuroleptic drugs are indicated in psychiatric patients. A central anticholinergic syndrome in the early postoperative period causative of the symptoms of delirium may respond to intravenous injection of physostigmine. Most of the time, however, these acute disturbances of brain function are best treated by correction of homeostatic imbalances, restoration of cardiovascular and respiratory stability and alleviation of pain. Postoperative delirium occurring two or more days later is frequently due to respiratory distress, followed by sepsis, alcohol withdrawal and many other causes including heart failure, exsiccosis and side-effects of drugs. In intensive care patients, delirium may be caused, for example, by withdrawal (alcohol, opioids, benzodiazepines), the onset of sepsis (often venous catheter related), side-effects of drugs, problems of communication, sleep deprivation and others. Treatment should focus on finding the right approach. Personal care should be intensified and include help from family members. Most problems arise from agitated, non-cooperative patients. Treatment with clonidine, gamma-hydroxybutyric acid or neuroleptic drugs like perazin and haloperidol may be required to reduce agitation and the activation of sympathetic influence.

Critical Care↗
PubMed2002

Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water.

Biological membranes are often poorly visible with the electron microscope after high-pressure freezing and freeze-substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high-pressure frozen yeast cells, rat-pancreas tissue and arthropod tissue were freeze-substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1-5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze-substitution with water-free medium but good after addition of 5% water to the substitution medium.

Animals↗
PubMed2001

Liposomes as protective agents of stratum corneum against octyl glucoside: a study based on high-resolution, low-temperature scanning electron microscopy.

The ability of phosphatidylcholine (PC) liposomes to protect pig stratum corneum (SC) against the action of the nonionic surfactant octyl glucoside (OG) was investigated "in vitro" using double-layer coating for high-resolution, low-temperature scanning electron microscopy. This technique has been useful in preventing drying artifacts in the study of biological materials. The treatment of SC with OG led to a perturbation mainly in the corneocytes. However, the incubation of the tissue with liposomes prior to the OG treatment resulted in a progressive decrease in these perturbations and, consequently, in the progressive protection of the SC against the action of the surfactant.

Animals↗
PubMed2001

Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells.

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2+-concentration ([Ca2+]i) along their anterior-posterior axis involved in cell-type specific differentiation. [Ca2+]i is high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/min x mg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5vs 7.7 nmol/10(8)cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior-posterior [Ca2+]i-gradient.

Animals↗
PubMed2000

Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration.

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphibia). The axolotl system is an accepted model for studying mechanisms of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo. However, when tested on different alternating substrates in vitro, migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled "honeycomb"-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.

Ambystoma↗
PubMed1999

Electron microscopic investigation of water occlusions in intercellular spaces in the inner cortex of lucerne nodules.

It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne (Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.

Cryoelectron Microscopy↗
PubMed1999

Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning.

Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM. Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing. Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face. The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces. The cytoplasms show particles of different size which most likely represent proteins. The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging. High quality cryo-sections are only obtained from vitrified material. Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.

Animals↗
PubMed1997

Double-layer coating for field-emission cryo-scanning electron microscopy--present state and applications.

Imaging of fast-frozen samples is the most direct approach for electron microscopy of organic material. It prevents chemical fixation and drying artifacts. Frozen samples can be replicated and imaged in the transmission electron microscope (TEM), or they can be directly visualized in the cryo-scanning electron microscope (cryo-SEM). Double-layer coating combines these two techniques and many of their advantages. With this method, the frozen bulk sample is coated similar to the TEM-replica technique with, for example, a shadow of platinum (at an angle of 45 degrees) and an additional layer of carbon. Then, the sample is cryo-transferred to an SEM equipped with a cold stage and imaged with the material-dependent backscattered electron signal that shows the platinum distribution. With this method, charging artifacts and the effects of beam damage are significantly reduced. Although currently the resolution of the replica technique cannot be surpassed, the method greatly facilitates the processing of brittle, rapidly frozen samples because no replica cleaning is necessary. This makes the method especially suitable for high-pressure frozen samples.

Freeze Fracturing↗
PubMed1997

IGF-I and bFGF differentially influence atrial natriuretic factor and alpha-smooth muscle actin expression in cultured atrial compared to ventricular adult rat cardiomyocytes.

In the present study, we compare expression, storage and secretion of the atrial natriuretic factor (ANF) in atrial and ventricular adult rat cardiomyocytes (aARC and vARC) in long-term culture. The influence of insulin-like growth factor-I (IGF-I) and of basic fibroblast growth factor (bFGF) on ANF production and secretion, as well as on the expression of a structural component, alpha-smooth muscle actin (alpha-sm actin), was studied in the two cell types. Antibodies against alpha-ANF were used for immunocytochemical localization of ANF. aARC contained more ANF-granules than vARC, and they were distributed throughout the cell bodies. Quantitative determination of ANF storage and secretion was done by radioimmunoassay (RIA; 125I), and it was demonstrated that aARC stored and secreted ANF 18- and 16-times more, respectively, when compared to vARC. Immuno-electron microscopy confirmed that ANF storing secretory granules were present in both types of cardiomyocytes. Expression of ANF and alpha-sm actin in aARC and vARC responded differently to treatment with either IGF-I or bFGF. In aARC, neither IGF-I nor bFGF had an influence on expression of ANF. In vARC, expression of ANF was downregulated by IGF-I and upregulated by bFGF with regard to both immunoreactivity and message. In contrast to vARC, expression of alpha-sm actin was not affected by IGF-I in aARC, whereas bFGF produced a strong upregulation similar to that found in vARC. Mitogen-activated protein kinases (MAPK) 42 and 44, though, were equally activated by bFGF and IGF-I in both aARC and vARC.

Actins↗
PubMed1996

Light damage in rod outer segments: the effects of fixation on ultrastructural alterations.

PURPOSE: In this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina. METHODS: The alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures. RESULTS: Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar. CONCLUSIONS: We conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation.

Animals↗
PubMed1995

Double-layer coating for high-resolution low-temperature scanning electron microscopy.

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g., 100,000 x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e-nm-2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5-10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g., 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.

Carbon↗
PubMed1995

Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation.

High-pressure freezing-freeze substitution (HPF-FS) methods were applied to germinating pea leaves. Good ultrastructural preservation without visible freezing damage was obtained up to 200 microns in thickness. Compared to conventional chemical fixation (CF), cellular membranes were smoother without undulation, and organelles appeared more turgid. The matrices of cytoplasm and organelles were denser and more homogeneous. The features imply that HPF-FS samples retain more substances and ultrastructure closer to the living state. There were differences in membrane stainability among organelles in HPF-FS specimens, which were not seen after CF. Bundles of microfilaments were observed frequently after HPF-FS.

Freeze Substitution↗
PubMed1994

Immortalization of rat hepatocytes by fusion with hepatoma cells. I. Cloning of a hepatocytoma cell line with bile canaliculi.

Hepatocytoma (HPCT) hybrid cells were obtained by fusion of cultured rat hepatocytes with Fao Reuber hepatoma cells H35 by polyethylene glycol treatment. Surviving cells were cloned in HAT (hypoxanthine-aminopterine-thymidine)/ouabain medium and propagated in cell lines over 80 passages. Morphological criteria were chosen to allow differentiation of the clones into two types of cells: 1) Type I cells which formed irregular cell layers, lacked contact inhibition and resembled the parental Fao hepatoma cells and 2) type II cells, which proliferated in monolayer cultures, exhibited contact inhibition during growth in culture plates and formed bile canaliculi thereby resembling cultured hepatocytes by phenotype. Bile canaliculi were absent in type I clones and Fao cells. One particular type II clone 1E3 was studied in detail. These cells formed bile canaliculi sealed by tight junctions and were comparably polarized as cultured hepatocytes. They expressed canalicular antigen B10, canalicular aminopeptidase N, and even secreted the fluorescent bile acid derivative NBD-cholate into the canalicular lumen. This type of HPCT cells lacked malignancy by tests in vivo and in vitro, and contained 110 +/- 5 chromosomes. The cells were considered to represent an immortalized hepatocyte-like cell line.

Animals↗
PubMed1993

Heterogenous distribution of glycoconjugates in the kidney of dogfish Scyliorhinus caniculus (L.) with reference to changes in the glycosylation pattern during ontogenetic development of the nephron.

Eight fluorochrome-coupled lectins with different sugar specificities were applied to cryosections of dogfish kidney. Despite profound differences in renal architecture between elasmobranch fish and other vertebrates, the sequence of nephron segments as revealed by the lectin-binding pattern was rather similar to that of tetrapodes. Wheat germ agglutinin (WGA) bound to cell membranes of epithelial cells of glomeruli, proximal and distal tubules, their basement membranes, the collecting tubule, and epithelial cells. Among other broadly binding lectins were Ricinus communis agglutinin I (RCA-I), soybean agglutinin (SBA), peanut agglutinin (PNA), Lycopersicon esculentum agglutinin (LEA), and Jacalin, all of which marked proximal as well as distal portions of the renal tubule. Dolichos biflorus agglutinin (DBA) did not react with any renal structure. Ulex europaeus agglutinin I (UEA-I), which indicates the presence of alpha-L-fucose, very strongly and specifically marked single epithelial cells of the early distal nephron, all epithelial cells of the late distal tubule, the beginning of the collecting tubule in the mesial tissue zone, and single cells in the end portion of the collecting tubule in the lateral bundles. Binding of UEA-I to receptors of distal nephron cells could be useful for the identification of these cells in functional studies employing teased tubule and/or isolated cell preparations. Binding of UEA-I to dogfish kidney structures resembles staining with UEA-I conjugates of late distal tubules and collecting tubules in the kidneys of frog and other, higher vertebrates. Epithelial cells of early developmental stages showed, very rarely, binding sites for most lectin-fluorochrome conjugates. A large number of lectin binding sites was observed in the extracellular matrix of fibroblast layers surrounding the early anlage and the S-shaped body. Lectin binding sites of the nephron epithelia appeared in a sequential manner in the next stages of development of the nephron. Ontogenetic and phylogenetic aspects of the merging region between nephron proper (late distal tubule) and collecting system (collecting tubule) are discussed.

Animals↗