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Biomedical subjects

M Marko

Publications and source records attributed to M Marko.

At least 19 recordsLinked to original sources
PubMed2006

Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples.

The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30-keV Ga(+) beam, and cryo-transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200-150 nm, changes characteristic of heat-induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that 'cryo-FIB thinning' of bulk frozen-hydratred material will be capable of producing specimens for TEM cryo-tomography with much greater efficiency than cryo-ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.

Cryoultramicrotomy↗
PubMed2004

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ.

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double-tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron-opaque particles in the proximal axoneme which may represent intraciliary-transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.

Animals↗
PubMed2004

Synthesis and antifungal properties of compounds which target the alpha-aminoadipate pathway.

Fungi synthesize lysine via the alpha-aminoadipate pathway, which is not found in plants or animals. This pathway has been proposed as a target for antifungal agents, but until now no reports have appeared to test this proposal. Hampering studies on the susceptibility of filamentous fungi such as those of the clinically important genus Aspergillus is the fact that growth quantitation is notoriously difficult. We have used the recently-reported XTT-based method of biomass quantitation to measure the susceptibility of Aspergillus nidulans strain A28 to growth suppression by novel compounds designed to target early steps in the alpha-aminoadipate lysine biosynthesis pathway, specifically those steps involving (R)-homocitrate and (2R,3S)-homoisocitrate. Three compounds show moderate inhibition of fungal growth, which can be partially restored by the presence of lysine in the growth medium.

2-Aminoadipic Acid↗
PubMed2003

Effect of head-down bed rest on the neuroendocrine response to orthostatic stress in physically fit men.

The role of neuroendocrine responsiveness in the development of orthostatic intolerance after bed rest was studied in physically fit subjects. Head-down bed-rest (HDBR, -6 degrees, 4 days) was performed in 15 men after 6 weeks of aerobic training. The standing test was performed before, after training and on day 4 of the HDBR. Orthostatic intolerance was observed in one subject before and after training. The blood pressure response after training was enhanced (mean BP increments 18+/-2 vs. 13+/- 2 mm Hg, p<0.05, means +/- S.E.M.), although noradrenaline response was diminished (1.38+/-0.18 vs. 2.76+/-0.25 mol.l(-1), p<0.01). Orthostatic intolerance after HDBR was observed in 10 subjects, the BP response was blunted, and noradrenaline as well as plasma renin activity (PRA) responses were augmented (NA 3.10+/-0.33 mol.l(-1), p<0.001; PRA 2.98+/-1.12 vs. 0.85+/-0.15 ng.ml(-1), p<0.05). Plasma noradrenaline, adrenaline and aldosterone responses in orthostatic intolerant subjects were similar to the tolerant group. We conclude that six weeks of training attenuated the sympathetic response to standing and had no effect on the orthostatic tolerance. In orthostatic intolerance the BP response induced by subsequent HDBR was absent despite an enhanced sympathetic response.

Adult↗
PubMed2002

Electron tomography of frozen-hydrated isolated triad junctions.

Cryoelectron microscopy and tomography have been applied for the first time to isolated, frozen-hydrated skeletal muscle triad junctions (triads) and terminal cisternae (TC) vesicles derived from sarcoplasmic reticulum. Isolated triads were selected on the basis of their appearance as two spherical TC vesicles attached to opposite sides of a flattened vesicle derived from a transverse tubule (TT). Foot structures (ryanodine receptors) were resolved within the gap between the TC vesicles and TT vesicles, and some residual ordering of the receptors into arrays was apparent. Organized dense layers, apparently containing the calcium-binding protein calsequestrin, were found in the lumen of TC vesicles underlying the foot structures. The lamellar regions did not directly contact the sarcoplasmic reticulum membrane, thereby creating an approximately 5-nm-thick zone that potentially constitutes a subcompartment for achieving locally elevated [Ca(2+) ] in the immediate vicinity of the Ca(2+)-conducting ryanodine receptors. The lumen of the TT vesicles contained globular mass densities of unknown origin, some of which form cross-bridges that may be responsible for the flattened appearance of the transverse tubules when viewed in cross-section. The spatial relationships among the TT membrane, ryanodine receptors, and calsequestrin-containing assemblage are revealed under conditions that do not use dehydration, heavy-metal staining, or chemical fixation, thus exemplifying the potential of cryoelectron microscopy and tomography to reveal structural detail of complex subcellular structures.

Animals↗
PubMed2002

Effect of simulated microgravity on endocrine response to insulin-induced hypoglycemia in physically fit men.

Adaptation to microgravity is associated with alteration in some endocrine functions. In the present longitudinal study, the counterregulatory hormonal response to insulin-induced hypoglycemia (ITT, 0.1 IU/kg short acting insulin i. v.) was evaluated under simulated microgravity conditions in 15 physically fit subjects. ITT was performed at the beginning of the investigation, and again after completion of 6 weeks of endurance training and after a subsequent period of 4 days of head-down bed rest at a backward tilt of 6 degrees from the horizontal. Endurance training showed a significant increase in maximal aerobic capacity in previously well-trained subjects (increase by 12 %), as well as on attenuation of counterregulatory response of epinephrine to hypoglycemia. After 4 days of bed rest, basal concentrations of plasma norepinephrine was diminished (p < 0.002) and plasma renin activity was enhanced (p < 0.02). After bed rest, decreased responses of the two catecholamines (norepinephrine, p < 0.001; epinephrine, p < 0.001), growth hormone (p < 0.001), and cortisol (p < 0.05) were observed. Response of plasma renin activity after bed rest was increased (p < 0.01). This longitudinal study indicated that 4 days of bed rest in endurance-trained subjects induced increased response of PRA to hypoglycemia and attenuation of other counterregulatory neuroendocrine responses.

Adult↗
PubMed2002

Responses of sympathoadrenal and renin angiotensin systems to stress stimuli in humans during real and simulated microgravity.

Changes of plasma hormone levels were investigated in human subjects after exposure to physical exercise (WL) and insulin induced hypoglycemia (ITT) during space flight or after head down bed rest (HDBR). Exaggerated responses of plasma epinephrine (EPI), norepinephrine (NE) and aldosterone (ALD) were observed after WL during space flight as compared to preflight response. Hypoglycemia during space flight induced attenuated responses of EPI, NE and augmented response of ALD. Exposure to WL during HDBR was followed by significantly exaggerated responses of plasma EPI, NE, ALD, PRA and cortisol. In HDBR the responses of plasma EPI, NE and cortisol were reduced and PRA response was exaggerated during ITT. These data indicate that hormonal responses to ITT and WL are similar at real and simulated microgravity.

Comparative Study↗
PubMed2001

The emergence of electron tomography as an important tool for investigating cellular ultrastructure.

Electron tomography has emerged as the leading method for the study of three-dimensional (3D) ultrastructure in the 5-20-nm resolution range. It is ideally suited for studying cell organelles, subcellular assemblies and, in some cases, whole cells. Tomography occupies a place in 3D biological electron microscopy between the work now being done at near-atomic resolution on isolated macromolecules or 2D protein arrays and traditional serial-section reconstructions of whole cells and tissue specimens. Tomography complements serial-section reconstruction by providing higher resolution in the depth dimension, whereas serial-section reconstruction is better able to trace continuity over long distances throughout the depth of a cell. The two techniques can be combined with good results for favorable specimens. Tomography also complements 3D macromolecular studies by offering sufficient resolution to locate the macromolecular complexes in their cellular context. The technology has matured to the point at which application of electron tomography to specimens in plastic sections is routine, and new developments to overcome limitations due to beam exposure and specimen geometry promise to further improve its capabilities. In this review we give a brief description of the methodology and a summary of the new insights gained in a few representative applications.(J Histochem Cytochem 49:553-563, 2001)

Animals↗
PubMed2001

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

Antigens, CD↗
PubMed2000

Defects in TGF-beta signaling overcome senescence of mouse keratinocytes expressing v-Ha-ras.

Previous studies have shown that TGFbeta1 expression is upregulated in mouse keratinocytes infected with a v-rasHa retrovirus, although the functional significance of this has not been clear. Here we show that v-rasHa retrovirus transduced primary mouse keratinocytes undergo hyperproliferation followed by a TGFbeta1 dependent G1 growth arrest and senescence. The growth arrest is accompanied by a 15-fold increase in total TGFbeta1 secreted and a fourfold increase in secreted active TGFbeta1. When cultured in the presence of a neutralizing antibody to TGFbeta1, the senescence response is suppressed. Levels of the TGFbeta1 target p15ink4b increase during senescence as does association of this kinase inhibitor with cyclinD/cdk4 complexes. However, p16ink4a, p53 and p19ARF expression also increase during senescence. Genetic analysis shows that TGFbeta1 null and dominant negative TbetaBRII expressing v-rasHa keratinocytes resist the G1 growth arrest and do not senescence. This resistance is associated with low expression of p15ink4b and p16ink4a, constitutive Rb phosphorylation and high levels of cdk4 and cdk2 kinase activity. In contrast, inactivation of TGFbetabeta1 secretion or response does not block the induction of p53 and p19ARF, but the level of p21waf1, a p53 target gene, is reduced in cyclin D/cdk4 and cyclin E/cdk2 complexes. Thus, although multiple senescence pathways are activated in response to a ras oncogene, inactivation of TGFbeta1 secretion or response is sufficient to block the senescence program. Since v-rasHa transduced TGFbeta1-/- keratinocytes form squamous cell carcinomas following skin grafting, these results suggest that in mouse keratinocytes, defects in TGFbeta1 signaling accelerate malignant progression by overcoming oncogene induced replicative senescence.

Animals↗
PubMed2000

Health risk assessment for inhalation exposure to arsenic.

Health risk assessment was used as the formal process to estimate the likelihood and magnitude of the health effects occurring in humans as a result of environmental and occupational exposure to polluting agents. This study was focused at estimating the human health risk of the general and working population living in the region polluted by arsenic for more than 40 years, from combustion of coal with high arsenic content in the power plant. The exposure to arsenic from inhalation was under investigation. A study period of 40 years (1973-1993) was chosen. The study period was defined taking into account, besides the availability of data, the temporal patterns of the technological processes and the trends over time of environmental concentrations. The results from the arsenic risk assessment study were used for the evaluation of the health risk for different population groups in the polluted areas and for different professions of workers exposed to As in a power plant. The results are applicable for the evaluation of risk in real conditions, for health surveillance and for remedial changes and a potential suggestion on technological improvement.

Adult↗
PubMed2000

Insulin, catecholamines, glucose and antioxidant enzymes in oxidative damage during different loads in healthy humans.

Exercise, insulin-induced hypoglycemia and oral glucose loads (50 g and 100 g) were used to compare the production of malondialdehyde and the activity of antioxidant enzymes in healthy subjects. Twenty male volunteers participated in the study. Exercise consisted of three consecutive work loads on a bicycle ergometer of graded intensity (1.5, 2.0, and 2.5 W/kg, 6 min each). Hypoglycemia was induced by insulin (Actrapid MC Novo, 0.1 IU/kg, i.v.). Oral administration of 50 g and 100 g of glucose was given to elevate plasma glucose. The activity of superoxide dismutase (SOD) was determined in red blood cells, whereas glutathione peroxidase (GSH-Px) activity was measured in whole blood. The concentration of malondialdehyde (MDA) was determined by HPLC, catecholamines were assessed radioenzymatically and glucose was measured by the glucose-oxidase method. Exercise increased MDA concentrations, GSH-Px and SOD activities as well as plasma noradrenaline and adrenaline levels. Insulin hypoglycemia increased plasma adrenaline levels, but the concentrations of MDA and the activities of GSH-Px and SOD were decreased. Hyperglycemia increased plasma MDA concentrations, but the activities of GSH-Px and SOD were significantly higher after a larger dose of glucose only. Plasma catecholamines were unchanged. These results indicate that the transient increase of plasma catecholamine and insulin concentrations did not induce oxidative damage, while glucose already in the low dose was an important triggering factor for oxidative stress.

Adult↗
PubMed2000

Topical delivery of 13-cis-retinoic acid by inhalation up-regulates expression of rodent lung but not liver retinoic acid receptors.

Chemopreventive retinoids may be more effective if delivered to the lung epithelium by inhalation. 13-cis-Retinoic acid (13-cis-RA) was comparable to all-trans-retinoic acid (RA) in inducing transglutaminase II (TGase II) in cultured human cells. Inhaled 13-cis-RA had a significant stimulatory activity on TGase II in rat lung (P < 0.001) but not in liver tissue (P < 0.544). Furthermore, inhaled 13-cis-RA at daily deposited doses of 1.9 mg/kg/day up-regulated the expression of lung retinoic acid receptors (RARs) alpha, beta, and gamma at day 1 (RARalpha by 3.4-fold, RARbeta by 7.2-fold, and RARgamma by 9.7-fold) and at day 17 (RARalpha by 4.2-fold, RARbeta by 10.0-fold, and RARgamma by 12.9-fold). At a lower aerosol concentration, daily deposited doses of 0.6 mg/kg/day were also effective at 28 days. Lung RARalpha was induced by 4.7-fold, RARbeta by 8.0-fold, and RARgamma by 8.1-fold. Adjustment of dose by exposure duration was also effective; thus, inhalation of an aerosol concentration of 62.2 microg/liter, for durations from 5 to 240 min daily for 14 days, induced all RARs from 30.6- to 74-fold at the shortest exposure time. None of the animals exposed to 13-cis-RA aerosols showed RAR induction in livers. By contrast, a diet containing pharmacological RA (30 microg/g of diet) failed to induce RARs in SENCAR mouse lung, although it induced liver RARs (RARalpha, 21.8-fold; RARbeta, 13.5-fold; RARgamma, 12.5-fold); it also failed to induce lung TGase II. A striking increase of RARalpha expression was evident in the nuclei of hepatocytes. Pharmacological dietary RA stimulated RARalpha, RARbeta, and RARgamma as early as day 1 by 2-, 4-, and 2.1-fold, respectively, without effect on lung RARs. Therefore, 13-cis-RA delivered to lung tissue of rats is a potent stimulant of lung but not liver RARs. Conversely, dietary RA stimulates liver but not lung RARs. These data support the concept that epithelial delivery of chemopreventive retinoids to lung tissue is a more efficacious way to attain up-regulation of TGase II and the retinoid receptors and possibly to achieve chemoprevention.

Administration, Inhalation↗
PubMed1999

Cubic membrane structure in amoeba (Chaos carolinensis) mitochondria determined by electron microscopic tomography.

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae-matrix organization with mitochondrial activity.

Amoeba↗
PubMed1999

Three-dimensional transmission electron microscopy and its application to mitosis research.

Transmission electron microscopy produces images that are projections of the original object, with the consequence that features from different depths of the specimen overlap and give a confusing image. This problem is overcome by reconstructing the object in 3D from a series of 2D views using either serial thin section reconstruction or electron tomography. In the serial section approach, the series of 2D views is generated from images of successive serial sections cut thin enough to be effectively 2D slices of the specimen. For electron tomography the series of 2D views is generated by tilting a single, usually thicker, section in the electron beam. Resolution in the depth dimension is limited to twice the section thickness for serial section reconstruction and is determined by the number of tilt views collected (i.e., by the fineness of the angular interval between successive tilt views) for electron tomography. Both methods produce distorted 3D reconstructions because of missing material and alignment difficulties in the case of serial sections and the limited angular tilt range in the case of electron tomography. However, techniques have evolved for minimizing and circumventing these distortions and, as long as the user is aware of the limitations, misinterpretations can be avoided. Since electron tomography provides better resolution (generally 5-20 nm), it is the method of choice for determining detailed structural interactions such as the depth of kinetochore MT penetration into the kinetochore outer plate. On the other hand, serial section reconstruction is more effective for projects that require tracking through a complete object in the specimen, such as counting the number of kinetochore MTs on each kinetochore. If the project requires finding a relatively small object in a large specimen (e.g., finding centrioles in an oocyte), then it is sometimes advantageous to cut thicker plastic sections and analyze them via stereo viewing. The mitotic spindle, however, is generally too complex to be analyzed via stereo viewing. Currently, collapse of plastic sections in the electron beam limits the utility of serial section electron tomography. Once a 3D reconstruction is completed it must be analyzed with the 2D medium of the screen on a computer monitor. The easiest approach is usually to walk through the 3D reconstruction volume slice by slice. However, in order to appreciate 3D interactions, and to communicate the results to others, it is generally necessary to segment key components from the rest of the volume and use modeling and rendering techniques. Rendered surface views can easily be color coded and provided with a number of depth cues to simulate the surface viewing encountered in everyday life. In some instances, it is useful to look through a smaller portion of the reconstruction volume with "X-ray vision." This can accomplished by using volume rendering to create a series of semitransparent views from different tilt angles.

Animals↗
PubMed1999

Malondialdehyde, lipofuscin and activity of antioxidant enzymes during physical exercise in patients with essential hypertension.

DESIGN: To clarify the role of oxidative damage in essential hypertension, levels of lipid peroxidation products (malondialdehyde and lipofuscin) and activity of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were examined during a short period of physical exercise. PATIENTS AND METHODS: We studied 11 male patients with mild to moderate essential hypertension in World Health Organization classes I or II and 10 healthy male controls. Physical exercise was performed on a bicycle ergometer at graded intensities of 1.0, 1.5 and 2.0 W/kg body weight Plasma concentrations of lipofuscin, malondialdehyde, epinephrine, norepinephrine, insulin, free fatty acids and glucose were determined. Superoxide dismutase activity was analysed in erythrocytes and glutathione peroxidase activity in whole blood. RESULTS: Concentrations of lipofuscin and malondialdehyde were significantly elevated in hypertensive patients. Superoxide dismutase activity was not different between groups, while glutathione peroxidase activity was significantly decreased in hypertensive subjects. During exercise, the concentration of malondialdehyde and antioxidant enzyme activities increased significantly in both groups. No differences were found in absolute increases between the normotensive and hypertensive subjects. The levels of glucose, insulin and free fatty acids were similar in both groups. Basal concentrations of catecholamines and also the exercise-induced increases were lower in hypertensive patients. CONCLUSIONS: Our results indicate increased oxidative damage in patients with essential hypertension, which might be caused by a decrease in the activity of glutathione peroxidase. The ability of superoxide dismutase and glutathione peroxidase to respond to increased production of reactive oxygen species during a short period of physical exercise was not impaired in hypertensive subjects.

Adult↗
PubMed1998

Electron microscopic tomography of rat-liver mitochondria and their interaction with the endoplasmic reticulum.

Electron microscopic tomography can provide a complete three-dimensional range of information about subcellular structures. Applied to mitochondria, it has shown that the conventional textbook model of this organelle is incorrect. The infoldings of the inner membrane (called cristae) are connected by narrow tubular regions to each other and to the outside, suggesting that internal diffusion of ions, metabolites and proteins may be restricted. Tomographic reconstruction of mitochondria in situ indicates these organelles may occur in clusters with stacks of endoplasmic reticulum, forming extended structures that may be involved in [Ca++] regulation.

Animals↗