Activity of antioxidant enzymes during hyperglycemia and hypoglycemia in healthy subjects.
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Adult↗
Biomedical subjects
M Marko
Publications and source records attributed to M Marko.
Low-dose automated electron tomography: a recent implementation.
Low-dose automated tomography has been implemented on a 400-kV JEOL intermediate voltage electron microscope. Instrumentation and procedures for automatic tomographic series data collection are described. Difficulties encountered and ways to overcome them are discussed. A low-dose tomographic projection series of a triad junction of frog sartorius muscle was semiautomatically collected and a 3-D reconstruction of this organelle was made.
Animals↗
Reconsidering mitochondrial structure: new views of an old organelle.
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Animals↗
Creating a healthy work environment in the midst of organizational change and transition.
In the midst of organizational change and transition, the need for a healthy work environment is greater than ever. Leaders may be in a position of leading staff on a journey they would rather not be on. Although there may not be a choice of destination, there are many decisions to be made along the way that will impact the health and quality of the journey. Creation of a healthy work environment does not occur overnight. It requires acknowledgment of the reality of the present environment, clear behavioral expectations and standards, systems, and structures to ensure the organizational changes are enduring and a means to assess continually the health of the work environment. Leaders have an opportunity and a responsibility to structure organizations in such a way that dignity, integrity, honesty, and compassion are preserved.
Hospitals, University↗
Double-tilt electron tomography.
Fidelity of tomographic reconstructions is improved and reconstruction artifacts are reduced, without increasing the number of projections, by combining tilt series taken around two orthogonal axes. Test reconstructions were made from high-voltage EM of rat liver mitochondria in a 0.6 micron thick plastic section. A number of schemes for selecting tilt angles for the projections are compared. A new method for aligning fiducial markers is described. It uses an iterative algorithm to determine the shift, scale, in-plane rotation and tilt angle for each tilt image, enforcing agreement of the expected locations of the fiducial markers in 3D space. These 3D locations are used to find the orientation between two tilt series and to merge both sets of projections.
Animals↗
The internal compartmentation of rat-liver mitochondria: tomographic study using the high-voltage transmission electron microscope.
The three-dimensional organization of the internal compartments of conventionally fixed and embedded rat-liver mitochondria has been determined by tomographic reconstruction from tilt-series images collected on the Albany high-voltage electron microscope. The results indicate that the inner membranes of these organelles are predominantly tubular in the orthodox (expanded matrix) conformation, as previously suggested by scanning electron microscopy. In the condensed (contracted matrix) conformation, the intracristal space opens up into large irregularly shaped compartments which are connected to each other and to the external (intermembrane) space by tubes with approximately the same diameter (20 nm) as those observed in the orthodox state. These results raise several questions, in particular about the nature of the structural transitions that occur in the cristae during matrix expansion and contraction, and about the influence of inner-membrane shape on the diffusion of ions and metabolites between the intracristal and intermembrane compartments.
Animals↗
Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.
Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.
Animals↗
Cytoarchitecture of size-excluding compartments in living cells.
By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from otherwise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent non-excluding domains. The height of the distal excluding compartments was generally < or = 50% of the height in the adjacent non-excluding compartment. An electron-dense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin nonmuscle filamin (ABP280) and alpha-actinin. F-actin and ABP280, but not alpha-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the trans-Golgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.
3T3 Cells↗
Changes in Müller cell plasma membrane specializations during subretinal scar formation in the rabbit.
The aim of this study was to identify changes in Müller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of Müller cells. These processes transform from the slender, highly polarized structures seen in normal animals into irregular processes that form a glia limitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Müller cell structural and functional polarity during subretinal scar formation.
Animals↗
[Differentiation of the ruminal epithelium of cattle during intrauterine development].
The prenatal development of the bovine ruminal epithelium was studied with light- and electronmicroscopical techniques. During the period of the nonstratified epithelium a pseudostratified epithelium is found in the dorso-cranial part, whereas the other areas possess a one-layered epithelium, which is, like the pseudostratified epithelium, transformed to a multilayered epithelium from the 7th week onwards. From the 9th week the period of the stratified epithelium starts with the formation of the stratum profundum and stratum superficiale. First signs of keratinization are seen in the superficial cells from 2.3 months onwards. With 4 months fetal cornified cells can be identified, with 5.5 months a single-layered stratum basale is seen on the differentiating papillar connective tissue, and the superficial cells are transformed to balloon-cells. In suprapapillar areas, a stratum spinosum is formed at the prenatal age of 7.5 months. During epitheliogenesis a horizontal and vertical differentiation of the cells can be observed. The first one includes the differentiation of undifferentiated, embryonal cells to the basal cells of the stratum profundum, the latter the development of the basal cells to spinous cells and then to fetal cornified and balloon-cells. The ultrastructural changes during the process of keratinization were especially considered.
Animals↗
Implementation of a quality improvement program for a bedside blood glucose testing system: a collaborative endeavor.
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Blood Glucose Self-Monitoring↗
Confocal microscopy and three-dimensional reconstruction of electrophysiologically identified neurons in thick brain slices.
Three-dimensional morphology and electrophysiology were correlated from individual neurons in a thick brain slice preparation. The hippocampal formation from immature and adult rats was cut transverse to the longitudinal axis into 500 microns-thick slices which were maintained under physiologic conditions. Individual neurons were impaled and physiologically characterized using microelectrodes. Recordings were made from the soma and in some cases from a dendrite. The impaled neurons were filled through the microelectrode with the fluorescent dye lucifer yellow and imaged by confocal scanning laser microscopy using an analog preprocessor. As many as 180 optical sections were recorded as a function of depth through the slices. Images are presented as a series of optical sections, stereo pairs, or three-dimensional reconstructions. Both stereo contouring and volume rendering methods were employed, and the reconstructions were viewed from any arbitrary perspective. Dendritic and axonal fields were separated from each other and displayed separately or as different pseudocolors. The three-dimensional reconstructions provided perspectives that were difficult or impossible to appreciate by viewing the optical sections or conventionally formed stereo pairs.
Animals↗
The use of confocal microscopy and STERECON reconstructions in the analysis of sea urchin embryonic cell division.
A laser scanning confocal microscope has been used to investigate the development of the sea urchin embryo. The samples were fixed in Carnoy's solution at various developmental stages, stained for DNA with the Feulgen reaction, and optically sectioned with a BioRad MRC-500 confocal microscope. Computer-generated stereographic projection images and a three-dimensional contour tracing and reconstruction system were employed to investigate the cleavage pattern during the 6th cleavage division. Cell division is found to be asynchronous during the 6th cleavage, with macromere derivatives completing division first, followed by mesomeres, and finally by the outer quartet of micromeres (which begins division only after macromeres and mesomeres have completed their respective divisions). Sixth cleavage produces an embryo comprising 60 cells. Asynchronous division was also observed within individual tiers of blastomeres. Variations in the orientations of cell division axes within individual tiers of cells were also observed. The utility of computer-graphics reconstruction techniques for both quantitative and qualitative developmental analysis are discussed.
Animals↗
Organelle rearrangement and cell volume changes during squeezing invasion of peritoneal elastic lamina by targeted murine breast carcinoma cells.
Murine breast cancer cell lines were developed to selectively invade the peritoneum while they proliferated in ascites form in the abdominal cavity. In a dominant form of invasion, tumor cells showed special affinity for elastin fibers and squeezed through narrow gaps in the elastic fiber meshwork of the stroma. Even in fixed tissue, such cells could be recognized as being in the process of invasive migration because of their dumbbell shape. This appearance was similar to that of diapedetic blood cells traversing bone marrow sinus endothelium. Three-dimensional STERECON graphics reconstruction from serial thick sections of 44 such cells was carried out. The reconstructions showed that, in mid-penetration, the cells spread extensively over the exterior surface of the elastic fiber meshwork. The cell surface contact of these forward projections was mainly with the elastic fiber outer coat of microfibrils, but small areas of the cell surface also fused directly to inner-core elastin. The morphological rearrangement of the cytoskeleton was minimal in both types of attachment areas. The location of these forward facing attachments is consistent with mechanisms for pulling the invasive cell through the gap. Lamellopodia formation and clustering of cytoplasmic organelles occurred more commonly at the forward-facing part of the cell. Morphometry of the reconstructions showed that a contraction of the whole cell occurred during the squeezing/migration process suggestive of an additional pushing process. However, our invasive cell lines showed marked differences in the degree of cell shrinkage. The process of adhesion and squeezing of tumor cells through elastin meshworks in vivo is clearly a complex phenomenon. Changes in cell surface activity appear to play a significant role in establishing the necessary 'foothold' component of invasion and, possibly, in the generation of tractive force as well.
Animals↗
The relative merits of direct morphometry of reconstructions of whole cells, and statistical morphometry by stereology of random sections of cells.
Stereology, or the derivation of quantitative, three-dimensional (3-D) data about cells by statistical analysis of the structures of random sections, is widely used in cytology and pathology. However, there are situations where this approach is inadequate, and only an analysis of a homogeneous population of whole cells will give the required results. This involved 3-D reconstruction from physical or optical sections, or tomography or photogrammetry of whole-cell mounts. Use of stereo views of individual sections or projections adds considerably to the information available for both contouring and reconstruction. Recent image-processing advances in clinical radiography have shown, for the first time, that rapid, high-resolution digitization and contrast enhancement enable nearly all structural details to be routinely extracted from the micrographs and adequately portrayed. Three-D whole-cell reconstructions provide the digital data for many kinds of morphometric measurements on both whole cells and their individual organelles and membranes. Rapid fixation or freezing allows improved quantitative structure/function correlations of organelles with disturbances in cell metabolism or gene expression.
Animals↗
Targeting of peritoneum by the small numbers of isogeneic and allogeneic ascites carcinoma cells that infiltrate or attach to peritoneum during ascites growth.
In the course of development of an in vivo invasion model, sublines of a series of allogenic and isogeneic carcinoma cell lines have been selected that show enhanced invasion of the peritoneum. It was found that, during the proliferation of tumor cell lines in ascitic form in the abdominal cavity, small numbers of cells infiltrated or firmly adhered to the peritoneum in at least 8/12 of the tumor-host combinations tried. After thorough washing of the peritoneum it was disaggregated by an enzyme mixture, and the resulting mixture of normal and tumor cells was inoculated intraperitoneally. Peritoneal isolations were made serially for 3 to 12 times. In 6 of 8 cases where the isolation produced a stable ascites, the cells showed enhanced peritoneal invasion compared with the parent cell line. The invasion of some of the cell lines was tested in another invasion model consisting of cultured mouse buccal mucosa (9/10 cell lines invaded the explant). In 3/3 cell lines showing enhanced peritoneum invasion in vivo, there was no enhanced invasion of the buccal mucosa. The enhanced peritoneum invasion appears to be tissue specific rather than a general increase in invasion potential. Pairs of high- and low-invasive cell lines were obtained that should be useful for screening for invasion modulating agents using the mouse ascites/peritoneum in vivo model. It is suggested that the method might be generalized to produce various tumor cell lines that target for the normal tissues that are adjacent to proliferating solid or circulating tumors.
9,10-Dimethyl-1,2-benzanthracene↗
[Electrophysiological characterization of cell membranes of non-excitable transport epithelium using computer data processing].
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Animals↗
Three-dimensional reconstruction of cells from serial sections and whole-cell mounts using multilevel contouring of stereo micrographs.
A comprehensive computer-graphics-based system (STERECON) is described for tracing and digitizing contours from individual or stereopair electron micrographs. The contours are drawn in parallel planes within the micrographs. Provision is also made for tracing and digitizing in full three-dimensional (3-D) coordinates in any direction along linear structures such as cytoskeletal elements. The stereopair micrographs are viewed in combination with the contours being traced on a graphics terminal monitor. This is done either by projecting original electron micrograph (EM) negatives onto a screen and optically combining these images with contour lines being drawn on the monitor, or by first digitizing the images and displaying them directly on the monitor along with the contour lines. Prior image digitization allows computer enhancement of the structures to be contoured. Correction and alignment routines are included to deal with variable section thickness, section distortion and mass loss, variations in photography in the electron microscope, and terminal screen curvature when combining projected images with contour lines on the monitor. The STERECON system organizes and displays the digitized data from successive sections as a 3-D reconstruction. Reconstructions can be viewed in any orientation as contour stacks with hidden lines removed; as wire-frame models; or as shaded, solid models with variable lighting, transparency, and reflectivity. Volumes and surface areas of the reconstructed objects can be determined. Particular attention was paid to making the system convenient for the biological user. Users are given a choice of three different stereo-viewing methods.
Animals↗