Knockdown of peroxisome proliferator-activated receptor-beta induces less differentiation and enhances cell-fibronectin adhesion of colon cancer cells.

The role of peroxisome proliferator-activated receptor-beta/delta (PPAR-beta/delta) in the pathogenesis of colon cancer remains highly controversial. This study specifically silenced the PPAR-beta expression in three colon cancer cell lines with different metastatic potentials. Although PPAR-beta knockdown resulted in more malignant morphological changes, bigger colony sizes and lower carcinoembryonic antigen (CEA) secretion, and enhanced the cell-fibronectin adhesion, cell invasion and migration were unaffected. These effects were stronger in poorly metastatic cell lines compared with highly metastatic ones. Simultaneously, PPAR-beta knockdown decreased the mRNAs encoding adipocyte differentiation-related protein and liver fatty acid binding protein, and increased the mRNA of ILK, whereas the mRNAs encoding integrin-beta1 and angiopoietin-like 4 were unchanged. Using immunohistochemistry, we determined that the intensity of PPAR-beta expression was stronger in rectal cancers with better differentiation than in those with poor differentiation, and was stronger in early-stage tumors than in advanced ones. Together, these findings consistently indicate that PPAR-beta may facilitate differentiation and inhibit the cell-fibronectin adhesion of colon cancer, having a role as an inhibitor in the carcinogenesis and progression of colorectal cancer. Interestingly, PPAR-beta seems to have a more important role in poorly metastatic cells than in highly metastatic ones.

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.

Cryosputtering--a combined freeze-drying and sputtering method for high-resolution electron microscopy.

Preparing cellular structures for visualization by high-resolution scanning electron microscopy (SEM) is a multi-step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high-resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze-dryer to accommodate a magnetron sputtering head, in order to sputter-coat the frozen-dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1.5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent-extracted human glioma cells or fibroblasts and the specimens were examined by high-resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two-stage rotary vane pump was connected to the drying-coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at -90 degrees C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat formed during sputtering.

Problems associated with the preparation of whole mounts of cytoskeletons for high resolution electron microscopy.

The resolution currently available in both transmission and scanning electron microscopes is theoretically adequate to visualize the organization of the cytoskeleton at the supramolecular and macromolecular levels. However, achieving this resolution in practice requires that the methods used to prepare the specimens both preserve the structures of interest and render them visible for observation in the microscope without obscuring or altering them. In this paper we discuss our own and others efforts to develop methods to overcome several problems associated with preparing whole mounts of cytoskeletons for observation by electron microscopy. These problems include: controlling the degree to which cellular components are extracted; the effects of osmium tetroxide on the cytoskeleton; controlling and recognizing shrinkage and drying artifacts; the choice of a method of visualization; deposition of grain-free ultrathin films of metal; and interpreting the results. The standard procedure which we currently use consists of the following steps: growing cells on carbon-stabilized Formvar-coated gold electron microscope grids; extracting in 0.5% Triton X-100 detergent in a microtubule stabilizing buffer; postfixing in 2.5% glutaraldehyde in stabilizing buffer; freeze-drying; magnetron sputter-coating with 1.5 nm of tungsten; and observation by TEM, SEM, or STEM. Cytoskeletons prepared in this manner contain over 100 polypeptides and are composed of a complex three dimensional meshwork of clean, uniform filaments, the smallest of which are 7 nm in diameter. A structure resembling the microtrabecular lattice is present only if the cells are prefixed with a relatively long bifunctional protein crosslinking reagent prior to extraction with detergent.

Preparation of cytoskeletons of cells in culture for high resolution scanning and scanning transmission electron microscopy.

In this tutorial we describe our methods for preparing detergent-extracted cytoskeletons for observation by high resolution scanning (SEM) and scanning transmission (STEM) electron microscopy. We both discuss the theoretical background and provide practical procedures for each of the following steps: cell culture on Formvar-coated gold grids; prefixation with aldehydes or protein crosslinking reagents (homobifunctional N-hydroxy-succinimide esters); extraction with Triton X-100 or Brij 58 detergent in microtubule stabilizing buffer; postfixation in formaldehyde and glutaraldehyde; dehydration; critical point and freeze drying; sputter coating with 1-2 nm of platinum or tungsten; and examination by SEM and both normal and inverted contrast STEM. These methods produce cytoskeletal preparations in which filaments as fine as 7 nm are preserved and can be observed by scanning electron microscopy.

Comparison of the effects of critical point-drying and freeze-drying on cytoskeletons and microtubules.

We have compared the effects of critical point-drying (CPD) and freeze-drying (FD) on the morphology of Triton-resistant cytoskeletons and microtubules by scanning (SEM) and transmission electron microscopy (TEM). In general, cytoskeletons attached to Formvar films suffer less structural damage than cells or cytoskeletons attached to glass, because the Formvar film absorbs some of the stress associated with shrinkage during drying. However, as seen in stereo-pair electron micrographs, the three-dimensional structure of cytoskeletons prepared by FD is better preserved and shows fewer artefacts than those prepared by CPD. CPD specimens are flatter, often have a concave and apparently collapsed nuclear matrix and show large cracks both in the perinuclear zone and through the cytoskeleton. At least some of the damage appears to be due to residual water in the CO2 used as the substitution fluid, because cytoskeletons dried with a water filter attached to the CPD apparatus show substantially less damage than those dried without the filter. Freeze-dried cytoskeletons consist mostly of unbroken, smooth filaments and have no perinuclear open space. Comparison of the effects of drying on the diameters of in vitro polymerized microtubules showed that the diameter of microtubules is reduced after drying, but that FD causes significantly less shrinkage than CPD. Addition of 0.2% tannic acid to the glutaraldehyde fixative significantly reduces the shrinkage of CPD microtubules, but has no effect on FD microtubules. The observations on microtubules support the hypothesis that drying-induced shrinkage is the result of both pressure and solvent evaporation and they indicate that tannic acid stabilizes samples against the former but not the latter.

An experimental model system for leishmaniasis. An ultrastructural study on cultured macrophages exposed to Leishmania parasites and sodium stibogluconate.

To facilitate studies on the effect of chemotherapeutic agents on the host-parasite interaction in leishmaniasis, we have developed an experimental model for infecting mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigotes. As the drug action is often dependent on concentration, the distribution of sodium stibogluconate, which is the commonly used drug for treatment of leishmaniasis, was studied in various parts of the macrophages by energy dispersive X-ray microanalysis. The drug was found to accumulate in secondary lysosomes. The ultrastructural examination, using TEM and SEM, of macrophages, whose secondary lysosomes had been preloaded with gold particles, showed that leishmania parasites are phagocytosed and finally located in secondary lysosomes. Using flameless atomic absorption spectrophotometry, the concentration of Mn, Fe and Cu in promastigotes of Leishmania donovani, Leishmania aethiopica, Leishmania crithidia, Leishmania major and their culture media was estimated. Of the three transition metals, the parasites accumulated only Mn from the medium, which they may use in a primitive defense mechanism against reactive oxygen metabolites produced by macrophages during the respiratory burst associated with phagocytosis.

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy.

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

Characterization of monoclonal anti-DNA antibodies and visualization of binding to chromosomes and cell nuclei.

Monoclonal antibodies against DNA from two hybridoma cell lines were produced and characterized. One had specificity for single stranded (ss) DNA with some cross-reactivity to RNA, while the other was specific for both single (ss) and double stranded (ds) DNA. The latter ds and ss DNA-binding antibody was used as a model for analysing the distribution of the epitope in chromosomes and cell nuclei. A linear correlation between antibody binding and propidium iodide counterstaining was found on flow cytometric analysis of suspended chromosomes. Immunofluorescence of rat myoblast cells showed a speckled distribution of the antibody in the nucleus with a variability between the cells. Using electron microscopy to visualize antibody binding with gold particles, codistribution with uranyl acetate staining of leucocytes was found. These results suggested that the antibody preferentially binds to condensed chromatin in cells and chromosomes.

Surface histamine receptors in rat peritoneal mast cells. Separation of receptor-bearing cells via binding to a histamine-protein conjugate, Cell-ect.

The Seragen Cell-ect Total Histamine KitTM was used to separate histamine receptor-bearing rat peritoneal mast cells from cells lacking these receptors. It was found that approximately 75% of the peritoneal mast cell population carried cell surface histamine receptors. The results further suggest that the mast cell histamine receptors present are mainly of an H1-type, as judged by the capacity of a specific H1-antagonist to reduce the histamine receptor-dependent cell adhesion. Moreover, an H1-agonist is less efficient in this respect and an H2-antagonist does not affect the cell adhesion at all. A possible functional role for these receptors, however, remains to be clarified.

Histomorphology of idiopathic and symptomatic popliteal cysts.

Thirty popliteal cysts, classified on the basis of clinical arthrographic findings as 12 idiopathic and 18 symptomatic lesions, were examined by light and scanning microscopy. The histopathology varied considerably and did not allow a valid distinction between the two types of cysts. Nevertheless, careful histopathologic examination of surgically removed popliteal cysts is advisable for exclusion of malignancy and for consideration of the presenting symptom as part of an inflammatory arthritic disorder.

The fine structure of "dividers" and "non-dividers" in phase II human glial cell cultures.

A method is described which allows a comparison in the transmission electron microscope (TEM) of cells with different remaining proliferative capacity from one and the same culture. The method takes advantage of a mini-cloning technique employing hapatotactic palladium islands in combination with micro-dissection and preparation for TEM of islands carrying various numbers of cells after 10 days in culture, when all miniclones have become density dependent growth inhibited. By means of this technique non-dividers were compared with miniclones of dividers composed of five to eight cells originating from single cells. Moreover, large, immotile cells without peripheral ruffling activity, known to be non-dividers, were compared with small, ruffling cells, known to be dividers, in the reflection-interference mode in sparse cultures of living cells, and in the TEM mode as whole cell preparations after critical point drying of cells cultured on formvar-coated, gold EM-grids. Non-dividers proved to contain a moderate number of residual bodies, well developed Golgi areas, and often branched or circular mitochondria; they were thinly spread over the substratum with many focal points of contact, and large areas of close apposition between cell and substratum.

Visualization of the peripheral weave of microfilaments in glia cells.

A peripheral weave of microfilaments is visualized in human glia cells. In this weave small numbers of microfilaments converge to structures in the cell edge. Similar assemblies of microfilaments seem to be attached to structures on the surface of microspikes. Together with filaments splaying from the paracrystalline arrangement in microspikes, these units make up the peripheral weave. The filaments of the weave come in close contact with each other and with filaments of internal actin fibres.

Transmission and scanning electron microscopy of whole glioma cells cultured in vitro.

Twenty-six lines of malignant glioma cells have been established in our laboratories. The cell lines have all been derived from low-differentiated human astrocytomas which did not differ in their histopathological classification. A representative selection of these lines was cultured on formvar-covered, carbon stabilized, gold, EM grids. After glutaraldehyde and osmium fixation, they were dehydrated, critical-point dried and studied as whole-cell preparations in the transmission electron microscopy (TEM) mode. The specimens were then coated with 10-nm of a gold/palladium alloy and the same cells identified and examined in the scanning electron microscopy (SEM) mode. This combined TEM and SEM method makes it possible to relate intracellular structures to surface details, such as ruffles, endocytotic vacuoles, filopodia and microvilli. The study also revealed a large spectrum of dissimilarities between the studied lines, which underlines the fact that no essential structural features of a tumor type can be drawn from studies on occasional lines of cells derived from it.

Electron probe X-ray microanalysis of residual bodies in aged cultured human glial cells.

Secondary lysosomes of the residual body type are frequent in nondividing cells from phase III cultures of human glial cells. These organelles have previously been shown to be analogous to lipofuscin granules of postmitotic cells in vivo. Most recent studies favor the assumption that residual bodies mainly result from incomplete degradation within the lysosomal vacuome of endogenous cellular components such as mitochondria and endoplasmic reticulum. Since iron occurs in several metalloenzymes produced by such organelles, it should then be possible to demonstrate accumulated iron within residual bodies. X-ray dispersive analysis of sectioned biological material is often hampered by diffusion and dissolution during preparation, as well as by too low a concentration of the elements. In this study we cultured glial cells on Formvar-coated gold grids and studied them unsectioned, after brief glutaraldehyde fixation and freeze-drying, in a transmission electron microscope at 100 kV in TEM and STEM mode. It was then possible to demonstrate iron in residual bodies of aged cells, presumably because the type of preparation utilized does not permit much dissolution.

The fine structure of growing human glia and glioma cells. Whole cell preparations.

Three lines of normal human glia cells and eight established lines of malignant glioma cells have been studied in the electron microscope (E.M.), using preparations of critical-point dried whole cells, sparsely grown on formvar-coated, E. M., gold grids. The malignant cell lines showed a very varied morphology, almost every line having its peculiarities as compared to the essentially identical normal glia lines. The major differences noted concerned the form of the leading lamellae, number of microspikes and the distribution of organelles such as secondary lysosomes and mitochondria. No single consistent finding made it possible to differentiate the glioma cells as a group from the glia cells in sparse cultures. The findings of this study show some of the individual glioma cell lines to have characteristic cell-surface structures. They were found to be identical with the findings in previous SEM studies, suggesting the peculiarities of the individual malignant glioma lines to be stable and retained, despite continual passage.

Scanning electron microscopy of acarus scabiei.

Details in the anatomy of sarcoptes scabiei var. hominis are described. Scanning electron microscopy is of help in illustrating this parasite which is commonly seen in clinical practice. The technique also provides the possibility to differentiate between various types of mite.