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Biomedical subjects

E Csonka

Publications and source records attributed to E Csonka.

At least 19 recordsLinked to original sources
PubMed2000

Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells.

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.

Animals↗
PubMed1999

Expression of membrane-bound and soluble cell adhesion molecules by human brain microvessel endothelial cells.

Expression of membrane-bound (mb) and soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-alpha (TNF-alpha) has been measured by enzyme-linked immunosorbent assay in cultured human brain microvessel endothelial cells. Both the mb and the s forms of VCAM-1 and ICAM-1 were upregulated by TNF-alpha; however, the stimulation of the s forms was delayed in time. When piracetam, a neuroprotective drug, was added to the tissue culture medium simultaneously with TNF-alpha, the expression of mbVCAM-1 and ICAM-1 was lowered. Differential upregulation of mb and s forms of adhesion molecules and a novel effect of piracetam have been demonstrated in human brain microvessel endothelial cell cultures.

Brain↗
PubMed1999

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

Animals↗
PubMed1998

Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4.

The inflammatory mediators, cytokines and complement proteins are believed to regulate the sequential events during the development of lesions secondary to ischaemia and reperfusion. The endothelial cell monolayer of the brain microvasculature is the critical interface between the blood-borne mediators and brain tissue. The involvement of these cells in complement production and regulation has not been well documented. In the present study, expression of complement proteins (C1 inhibitor, factor H, factor B, C4) by cultured endothelial cells obtained from human brain microvessels has been characterized. Interferon gamma upregulates the production of all the complement factors studied. Serine proteases, plasmin and miniplasmin induce the expression of C4, decrease the level of ELISA detectable C1 inhibitor, and do not affect the production of factors H and B. These data indicate that complement proteins are expressed locally by the brain microvessels, and may modulate the inflammatory responses of brain tissue.

Brain↗
PubMed1998

Perturbation of the integrity of the blood-brain barrier by fibrinolytic enzymes.

The action of fibrinolytic enzymes (plasmin, miniplasmin, neutrophil leukocyte elastase) on the blood-brain barrier is investigated. The binding and the effects of the fibrinolytic enzymes are studied in the first subcultivation of human brain capillary endothelial cells. 125I-labeled plasmin, miniplasmin and neutrophil leukocyte elastase bind to confluent monolayers of cultured endothelial cells with dissociation constants of 1 x 10(-8) mol/l, 4.8 x 10(-7) mol/l and 1.8 x 10(-8) mol/l, respectively, and the number of binding sites varies between 2.3 x 10(5) and 7.5 x 10(6) per cell. Following treatment of the cultured cells with purified and active-site titrated proteases, the changes in morphology of individual cells are analyzed with computerized morphometry. At low concentrations (in nanomolar range) all studied fibrinolytic proteases induce reduction of the cell area; the minimal size is achieved in 20-80 min after the application of an enzyme and the effect is completely reversed in 15 min after its removal. A possible in-vivo consequence of these in-vitro findings is studied in an organ-perfusion model: rat hemisphere is perfused with a protease solution followed by a circulating phase-borne tracer (horse-radish peroxidase). In perfused rat hemisphere, the fibrinolytic enzymes open the blood-brain barrier to the circulation-borne tracer. These results support the concept that fibrinolytic enzymes interact with the brain microvascular endothelium and thus affect the integrity of the blood-brain barrier through active cell contraction.

Blood-Brain Barrier↗
PubMed1996

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Animals↗
PubMed1996

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

Animals↗
PubMed1995

Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. An in vitro cell culture model.

BACKGROUND AND PURPOSE: Vasogenic brain edema is a frequent complication of ischemic stroke. The mechanism of the blood-brain barrier opening that underlies the edema formation is poorly understood. In the present study we examined the response of endothelial cells cultured from adult human brain to thrombogenic and fibrinolytic factors that possibly accumulate in the occluded vascular segments in ischemic stroke. METHODS: The changes in the morphology of cultured human brain microvascular endothelial cells were observed by phase-contrast light microscopy and quantified with computerized morphometry. RESULTS: Active proteases (eg, thrombin, plasmin, urokinase) as well as heparin and protamine, but not fibrinogen and antithrombin III, produced significant changes in endothelial cell morphology. Two shape patterns of contraction were observed: protamine treatment resulted in rounded cells with a decrease in both cell perimeter and area, whereas all other agents induced spiderlike cell morphology with increased perimeter and reduced area. The rate of contraction was dose dependent, and at comparable enzyme concentrations plasmin produced faster contraction than thrombin. The observed changes were reversed 3 hours after abrogating the treatment. CONCLUSIONS: In an in vitro model we have demonstrated that factors involved in thrombus formation and dissolution induce endothelial cell contraction, which could affect focally the permeability of the blood-brain barrier by opening paracellular avenues between endothelial cells in vivo. Thus, the genesis of brain edema in thromboembolic stroke or occasionally during fibrinolytic therapy can be attributed in part to the contact of these factors with the microvascular endothelium.

Brain↗
PubMed1990

Influence of the measles virus on the proliferation and protein synthesis of aortic endothelial and smooth muscle cells.

To clarify whether some viruses could influence the different functions and membrane permeability of the aortic cells, we have examined in a model experiment the in vitro effect of the measles virus on the aortic endothelial and smooth muscle cells. The aortic cells infected with the virus failed to reveal gross cytopathic effect. Occasionally, however, syncytium formation and nuclear inclusions were observed. In infected endothelial cells lysosome containing viral nucleocapsids were seen. The early phase of measles virus replication inhibited the proliferation of endothelial cells of all species tested, while uniformly stimulated the replication of the smooth muscle cells relative to the control. In bovine aortic endothelial and smooth muscle cells the protein synthesis had been suppressed by the 4th to 6th hours postinfection. The results indicate that measles virus infection may be among the risk factors of atherosclerosis. It may damage endothelial cells by altering the cell membrane permeability and could induce proliferation of aortic smooth muscle cells.

Animals↗
PubMed1987

Interaction of antithrombin III and thrombin-antithrombin III complex with cultured aortic endothelial cells.

The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3 X 10(-8) M and with 5 X 10(4) binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.

Animals↗
PubMed1987

Interspecific interaction of aortic endothelial and smooth muscle cells.

The proliferation stimulating or inhibitory effect of aortic endothelial cells on aortic smooth muscle cells and vice versa was studied in cell culture by using a newly developed co-cultivation technique. The effect was registered as the incorporation of labelled thymidine detected by liquid scintillation counting. The experiments were performed in intra and interspecific combinations, using bovine, pig or rat aortic cell cultures. The endothelial cells stimulated smooth muscle cell proliferation in a species specific way. In interspecies combinations either inhibition or no effect was observed. The smooth muscle cells usually had an inhibitory effect on the proliferation of endothelial cells except for the pig smooth muscle cells which had a stimulatory effect in both inter and intraspecies combinations. Co-cultivation of smooth muscle or endothelial cells with cells of the same type resulted mostly in inhibition or no effect in both intra and interspecies combinations. This phenomenon was particularly conspicuous with endothelial cells. The different stimulatory and inhibitory effects produced by these cells may play an important role in the regulation of neovascularization and in reendothelization of denuded intimal areas.

Animals↗
PubMed1986

Metabolism of non-enzymic glycosylated low density lipoprotein by mini pig aortic endothelial cells.

[U-14C]-glucose was incorporated non-enzymatically into the protein moiety of human low density lipoprotein. The incorporation was time and glucose concentration dependent. Investigated mini pig aortic endothelial cells showed that the glycosylated low density lipoprotein was bound, internalized and degraded significantly less than that of the native one measured with [125-I]-low density lipoprotein.

Animals↗
PubMed1986

Aortic endothelial and smooth muscle cell interactions in culture.

The effect of conditioned media on cell growth was studied in pig aortic smooth muscle cell conditioned medium on pig or bovine aortic endothelial cells and pig or bovine endothelial cells conditioned medium on pig aortic smooth muscle cell proliferation. Co-culturing experiments were also done in the same combinations. Cell proliferation was measured in both systems by 3H-thymidine incorporation autoradiography. In intraspecies combinations the media conditioned by confluent pig-aortic endothelial monolayer had an inhibitory effect on pig-aortic smooth muscle cell growth, but in the co-cultivation system the exponentially grown endothelial cells stimulated smooth muscle cell proliferation. The pig aortic smooth muscle cells had some stimulatory effect on pig endothelial cell proliferation in both systems. In the interspecies combination the media conditioned by confluent bovine aortic endothelial monolayer or, in the co-cultivation experiments, the bovine endothelial cells exerted a stimulatory effect on the proliferation of pig-aortic smooth muscle cells. The media conditioned by pig smooth muscle cells had also some little stimulatory effect on bovine aortic endothelial cell growth, but in the co-cultivation system the bovine aortic endothelial cells responded in a highly sensitive way to the stimulatory effect of pig aortic smooth muscle cells.

Animals↗
PubMed1985

The effect of nicotine on cultured cells of vascular origin.

Monolayers of endothelial, smooth muscle and fibroblastic cells of healthy porcine, bovine or human fetal origin were treated with 10(-4) to 10(-9) M final concentrations of nicotine. The effect was registered as changes in the synthesis and polyimerization of the cytoskeleton. The silver and gold impregnation method produced anisotropy of the synthetic granules and of the final polymers of microtubules and filaments under physiological conditions as revealed by polarization microscopy. Since the orientation of the cells was inhomogeneous in the cultures, the organization of the orientation was expressed as the sum of alternative diagonal and orthogonal measuring of anisotropy by a computerized microraster morphometry system joined to an OPTON cytophotometer. The 8-day-old control and treated cultures were also examined by electronmicroscopy. Nicotine stimulated the synthesis and polyimerization of the cytoskeletal protein. This phenomenon is evident in smooth muscle cells, and partly also in endothelium. Fibroblasts were not influenced by the doses of nicotine tested.

Animals↗
PubMed1984

Interaction of plasmin with endothelial cells.

Interaction of human plasmin with a monolayer culture of mini-pig aortic endothelial cells was studied by using the 125I-labelled enzyme. The binding of plasmin was time- and concentration-dependent. Equilibrium between bound and free enzyme was obtained within 90s, and Scatchard analysis indicated a high- and a low-affinity population of binding sites of approx. 1.24 X 10(4) sites/cell having a Kd of 1.4 X 10(-9) M and 7.2 X 10(4) sites/cell with a Kd of 2 X 10(-8) M respectively. Plasmin, bound to cell, was spontaneously released within 2 min, suggesting a rapid equilibrium. Chemical modification of the enzyme with phenylmethanesulphonyl fluoride or pyridoxal 5'-phosphate revealed that neither the active centre nor the heparin-binding site of plasmin was involved in the interaction with the endothelial cell. In terms of endothelial-cell receptors, the binding sites of cells for plasmin and thrombin were different: the two enzymes did not compete with each other, and the pretreatment of cells with neuraminidase or chondroitin ABC lyase resulted in a 50% decrease of thrombin or plasmin binding respectively. Arachidonic acid incorporated into phospholipids of the cell was released by plasmin, but a change in the rate of prostacyclin formation was not measurable. The interaction of plasmin with endothelial cells seems to be specific in the fibrinolytic system, since plasminogen did not bind to these cells under similar conditions.

Animals↗