Biomedical subjects

M Szabolcs

Publications and source records attributed to M Szabolcs.

At least 37 records · Page 2Linked to original sources

PubMed1996

Apoptosis of cardiac myocytes during cardiac allograft rejection. Relation to induction of nitric oxide synthase.

BACKGROUND: Apoptosis is a distinct form of programmed cell death characterized by activation of endonucleases that cleave nuclear DNA, condensation and fragmentation of nuclear chromatin, blebbing of intact membranes, and cell shrinkage and fragmentation. The mechanisms responsible are unclear, but nitric oxide (NO) generated by inducible NO synthase (iNOS) has been demonstrated to induce apoptosis in macrophages in vitro. This study investigated whether apoptosis occurs during cardiac allograft rejection and examined the relationship of apoptosis to iNOS expression. METHODS AND RESULTS: Heterotopic abdominal transplantation from Lewis to Wistar-Furth rats was used as a model of cardiac allograft rejection; Lewis-to-Lewis grafts served as controls. Apoptosis was identified by DNA ladders after electrophoresis on agarose gels and by in situ labeling of DNA fragments; cell types were determined by immunohistochemistry. The number of apoptotic cardiac myocytes increased sharply from day 3 (0.31/mm2 ventricular tissue) to day 5 (1.27/mm2) after transplantation. At day 5, allografts showed a significant increase (P < .01) in apoptotic cardiac myocytes, macrophages, and endothelial cells compared with syngeneic grafts. The expression of iNOS mRNA, protein, and enzyme activity paralleled in time and extent the apoptosis of cardiac myocytes. iNOS immunostaining of infiltrating macrophages and cardiac muscle fibers increased significantly in the allografts at days 3 to 5 and was accompanied by immunostaining of both cell types for nitrotyrosine, which is indicative of peroxynitrite formation. CONCLUSIONS: Apoptosis of myocardial cells occurs during cardiac allograft rejection. Apoptosis during rejection parallels the expression of iNOS, which suggests that apoptosis may be triggered by NO and peroxynitrite.

Animals↗

PubMed1996

Dysregulation of cellular proliferation and apoptosis mediates human autosomal dominant polycystic kidney disease (ADPKD).

The proto-oncogene c-myc has been implicated in both cellular proliferation and apoptosis, and we have shown that overexpression of c-myc can induce polycystic kidney disease in transgenic mice. To elucidate the molecular and cellular defects underlying cystogenesis, we have investigated the potential roles of cell proliferation and apoptosis as they relate to c-myc and modulators of c-myc function in human autosomal dominant polycystic kidney disease (ADPKD). Renal c-myc expression was consistently elevated, up to 15-fold, in ADPKD. High levels of c-myc expression correlated with 10- to 100-fold increased proliferation index in cystic epithelium. Interestingly, steady-state levels of bcl-2 mRNA were also increased up to 20-fold and Bcl-2 protein was markedly elevated. In contrast, the expression of bax and p53 was virtually unchanged. However, apoptosis was consistently and significantly increased in ADPKD kidneys, unchecked by high levels of Bcl-2. Together with proliferation, apoptosis may thus represent a general mechanism for cyst growth and tissue remodeling. We conclude that both epithelial cell proliferation and apoptosis required for normal kidney homeostasis are deregulated in ADPKD, recapitulating the renal developmental program. Furthermore, abnormal expression of proto-oncogenes regulating these processes is an important mediator of cystogenesis in human ADPKD.

Adult↗

PubMed1996

Oxidized low density lipoproteins alter macrophage lipid uptake, apoptosis, viability and nitric oxide synthesis.

Uptake of oxidized low density lipoproteins (LDL) by monocyte macrophages to form "foam" cells occurs during formation of atherosclerotic lesions. Inducible nitric oxide synthase (iNOS) has been identified in foam cells. To investigate interactions between oxidized LDL, monocyte macrophage viability and iNOS, studies were performed with J774.Al macrophages. iNOS mRNA, protein and enzyme activity were induced in J774.Al macrophages by IFN-gamma lipopolysaccharide (LPS). Neither iNOS induction nor inhibition of nitric oxide (NO) formation was associated with significant alterations in the binding, uptake or degradation of native or oxidized LDL. Nontoxic doses of native LDL or of oxidized LDL did not influence iNOS mRNA or protein in macrophages. However, oxidized LDL, but not native LDL or acetyl LDL, inhibited NO production by macrophages in a dose- and time-dependent fashion. Inhibition of iNOS was not correlated with cholesteryl ester formation but with the degree of LDL oxidation. Inhibition of iNOS did not require the scavenger receptor or directed endocytosis and exhibited noncompetitive kinetics. Inhibition of iNOS activity in J774.Al macrophages was produced by lipid from oxidized LDL but not by lipid from native LDL and by PC vesicles containing LPC but not by PC vesicles alone. Inhibition of NO formation diminished apoptosis of the activated macrophages. The data suggest NO production by iNOS and inhibition of the enzyme by oxidized LDL lipid may influence cell viability, cell-cell interactions and vasomotor tone during atherogenesis.

Animals↗

PubMed1995

Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals.

CD40 is expressed on a variety of cells, including B cells, monocytes, dendritic cells, and fibroblasts. CD40 interacts with CD40L, a 30-33-kD activation-induced CD4+ T cell surface molecule. CD40L-CD40 interactions are known to play key roles in B cell activation and differentiation in vitro and in vivo. We now report that normal human endothelial cells also express CD40 in situ, and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections from normal spleen, thyroid, skin, muscle, kidney, lung, or umbilical cord were studied for CD40 expression by immunohistochemistry. Endothelial cells from all tissues studied express CD40 in situ. Moreover, human umbilical vein endothelial cells (HUVEC) express CD40 in vitro, and recombinant interferon gamma induces HUVEC CD40 upregulation. CD40 expression on HUVEC is functionally significant because CD40L+ Jurkat T cells or CD40L+ 293 kidney cell transfectants, but not control cells, upregulate HUVEC CD54 (intercellular adhesion molecule-1), CD62E (E-selectin), and CD106 (vascular cell adhesion molecule-1) expression in vitro. Moreover, the kinetics of CD40L-, interleukin 1-, or tumor necrosis factor alpha-induced CD54, CD62E, and CD106 upregulation on HUVEC are similar. Finally, CD40L-CD40 interactions do not induce CD80, CD86, or major histocompatibility complex class II expression on HUVEC in vitro. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vitro. Moreover, they suggest a mechanism by which activated CD4+ T cells may augment inflammatory responses in vivo by upregulating the expression of endothelial cell surface adhesion molecules.

Antigens, CD↗

PubMed1994

Further characterization of the 3-dimensional crystals of detergent-solubilized (Na+,K+)-ATPase from pig kidney.

Multilamellar 3-dimensional (Type I) crystals of detergent-solubilized, purified (Na+, K+)-ATPase enzyme of pig kidney grow in media consisting of 0.1 M KCl, 0.1 M NaCl, 20 mM imidazole pH: 7.5 (20 degrees C), 5 mM MgCl2, 5 mM DTT, 3 mM ZNaN3, 0.025 TIU/ml Aprotinin, 2 micrograms/ml BHT and 20-40% glycerol, using nonionic detergents of C12E8 or BRIJ 36 for solubilization. The refined crystallization protocol: the use of media containing 20% glycerol, the low detergent: protein ratio and preincubation at subzero temperature at the initial phase of crystallization resulted in a remarkable increase of the yield and overall dimension of the crystals (up to 3-4 microns), while the stacking of the crystalline sheets was dramatically reduced. Biochemical and structural analysis of these crystals revealed further similarities between the 3-d crystals of the (Na+, K+)-ATPase and the Ca2+ ATPase of skeletal muscle-SR (Taylor and Varga, J. Biol. Chem., 269, 10107-10111, 1994). Computer image processing of the electron micrographs of stacked crystalline sheets of (Na+,K+)-ATPase molecules gave unit cell dimensions: a = 166.2 +/- 3.8 A, b = 54.2 +/- 3.5 A, with an included angle of 90 degrees C. Based on the close identity of the filtered images in projection and of other data, we concluded that the 3-dimensional crystals of the (Na+, K+)-ATPase contain only the alpha catalytic subunits.

Animals↗

PubMed1994

Enzymatic properties, metal composition and SH-group reactivity of the light and heavy sarcoplasmic reticulum vesicles.

Light (LT) and heavy (TTC) microsomes were isolated from the fragmented sarcoplasmic reticulum (FSR) of rabbit skeletal muscle by sucrose gradient centrifugation. The amount of the protein components (ATPase, "feet proteins", calsequestrine) showed substantial differences between the light and heavy fractions. The amounts of calcium, magnesium and zinc were about 2-4 times higher in the TTC fraction, then those of the LT fraction. The activities of the Ca2+ + Mg2+ activated para-nitrophenyl-phosphatase and acetylcholin-esterase were (about 1.5 times) also higher in the TTC fraction compared to the LT fraction. The ratio of the Ca-transport vs. ATP was 2.1 in case of the LT and 0.6 in case of the TTC fraction. The number of titerable SH-groups of the LT fraction, measured in EGTA-medium containing Ca2+, was higher than those measured in the absence of Ca2+, while for the TTC fraction this number was higher when determined in the absence of Ca2+. We suppose that due to the higher amount of Ca2+ and Zn2+ in the TTC fraction those SH-groups which were present as Ca- or Zn-thiolates became titerable in EGTA-medium in the absence of Ca2+.

4-Nitrophenylphosphatase↗

PubMed1994

Furosemide-induced lysosomal enzyme release in mouse kidney in vivo and in vitro.

The loop diuretic, furosemide, which has been known to elicit renin release in vivo as well as in vitro, has also been shown to induce lysosomal enzyme release. After administration of furosemide (10 mg/kg and 300 mg/kg), a significantly increased acid phosphatase activity (1.33 x 10(-4) and 1.73 x 10(-4) vs. 0.23 x 10(-4) U, p < 0.001) was detected in the urine of the drug-treated mice, accompanied by a reduction of the residual enzyme activity in the kidney (5.0% for 10 mg/kg and 18.4%--p < 0.05--for 300 mg/kg dosage). Two marker enzymes, acid phosphatase and beta-D-glucuronidase, were assayed to demonstrate that furosemide also exerts its effect in in vitro systems like renal cortex suspension (corr. coeff.: 0.899 for acid phosphatase and 0.908 for beta-D-glucuronidase, p < 0.001) and isolated lysosomes (corr. coeff.: 0.981 for acid phosphatase and 0.989 for beta-D-glucuronidase, p < 0.001 for both). This action of the drug seems to be different from the well-known furosemide-sensitive inhibition of ion transport systems and may become of clinical relevance for patients receiving high doses of furosemide.

Acid Phosphatase↗

PubMed1991

Secretory component of cystic craniopharyngiomas: a mucino-histochemical and electron-microscopic study.

Ten cases with cystic craniopharyngioma were investigated. Histologically, eight of them belonged to the adamantinomatous group and two were squamous epithelial type. Histochemical investigation revealed mucin secretion in microcysts, and electron microscopy demonstrated zymogen granules in the epithelial cells. When the protein content of the cyst fluid was analyzed by polyacrylamide-gel electrophoresis, the electrophoretic pattern and immunological properties were found to be similar to the normal human serum control. The results of the morphological study suggest that cystic craniopharyngiomas have a secretory component in addition to the classical histological structures.

Ameloblastoma↗

PubMed1990

Enzymatic properties, metal composition and SH-group reactivity of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle.

Comparison of properties of fragmented sarcoplasmic reticulum samples isolated by several methods is reported. It was found that the protein composition does not differ significantly in samples which were or were not washed with 0.6 M KCl when isolated. In the case of samples washed with KCl solution the Zn concentration, the Ca/Mg ratio (determined from experimental data), acetylcholinesterase and superoxide dismutase activities were higher whereas Ca+Mg-activated ATPase and p-nitrophenylphosphatase activities were lower than those of samples which were not washed with 0.6 M KCl. In the latter samples the amount of SH-groups and the reactivity of fast SH-s are higher in Ca+EGTA containing media than in media containing only EGTA. In contrast in the case of samples washed with KCl solution the results are the opposite. In conclusion, washing of FSR with 0.6 M KCl alters the metal composition, enzymatic properties, SH-group reactivity and as a result of these probably the conformation of the protein samples, as well.

4-Nitrophenylphosphatase↗

PubMed1989

Determination of the sedimentation coefficient and molecular weight of proteins by density gradient ultracentrifugation in fixed angle rotor.

The applicability of density gradient ultracentrifugation using fixed angle rotors for the determination of sedimentation coefficient and molecular weight of proteins was studied. Ovalbumin, bovine serum albumin, IgG-, IgA- and IgM-globulin proteins, as standards were, among others, centrifuged in a fixed angle rotor (Beckman type 50) in 5-20% (w/v) sucrose density gradient for 300 min, at a speed of 40,000 rpm, at 5 degrees C. After centrifugation, the sedimentation diagram of the above-mentioned proteins was taken by a spectrophotometer equipped by a flow-through cuvette and a compensator. The sedimentation path was measured on the diagrams. A calibration curve was drawn by plotting the sedimentation coefficients of the standard proteins against the sedimentation paths. This curve, for which the equation y = 1.74x-1.6 is valid, was used for the determination of the sedimentation coefficients of several globular proteins under the conditions described above. The values obtained this way agree within +3-10% with those determined with analytical ultracentrifuge. If the logarithms of proteins molecular weight used as standards were drawn against the logarithms of their sedimentation paths a calibration curve was obtained. This curve for which the equation ln y = 2.022 ln x + 8.71 is valid can be used for the determination of molecular weights of globular or nearly globular proteins.

Animals↗

PubMed1988

Isomyosin changes after functional electrostimulation of denervated sheep muscle.

Isomyosin analyses by biochemical, immunochemical, and histochemical investigations have been carried out in five sheep following unilateral recurrent laryngeal nerve paralysis and direct functional electrostimulation of the denervated cricoarytenoid posterior muscle. Myosin light chains were identified by two-dimensional gel electrophoresis. Myosin heavy chains were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis. Slow myosin heavy chain was identified by orthogonal peptide mapping and immunochemistry. The stimulation effect at cellular level was determined using adenosine triphosphatase (ATPase) histochemistry. A dramatic increase of the type 1 fiber area (slow, fatigue-resistant fibers) could be seen after many weeks of an increasing regime of low-frequency direct electrical stimulation. Biochemically, the amount of slow myosin was always higher than in normal muscles. Some muscles were transformed almost completely to the slow type. At the time they were studied and with the methods employed, the expression of embryonic isomyosin was not observed. In conclusion, after numerous weeks of maintained functional activity, elicited by direct electrostimulation, the denervated muscle regionally showed areas of hypertrophy or at least lack of atrophy of slow myofibers without major signs of muscle damage.

Animals↗

PubMed1988

Curing of 60Co gamma-resistance inducing effect of R46 R-factor by 5-fluorouracil.

The 60Co gamma-resistance inducing effect of R46 factor, and its elimination by 5-fluorouracil were studied. R46 increased the survival of the wild-type strain and its rec- mutants. After treatment with 5-fluorouracil (1 g/liter) the clones lost not only antibiotic resistance, but the 60Co gamma-radioresistance as well, encoded by R46 R-factor.

Escherichia coli↗

PubMed1987

In vitro cross-linking of gluten into high-molecular-weight polymers with transglutaminase.

From the amino acid composition of gluten proteins and the substrate specificity of transglutaminases (TGase) we concluded that gluten proteins can be favourable substrates for TGases due to their high glutamine content. By use of sodium dodecyl sulfate polyacrylamide gel-electrophoresis it was demonstrated that from gluten-ES and gluten-TS high-molecular-weight proteins developed in the presence of Ca2+ and red blood cell lysate containing TGase. When ovalbumin or deamidated gluten were applied as substrates no high-molecular-weight products were formed. Upon spectrophotometric measurements we found that covalent cross-links (isopeptide bonds) formed under the effect of TGases presumably cause a change in the position of chromophore groups in the substrates. Absorption decrease was detected between 274-276 nm as a result in the case of gluten-TS and gluten-ES used as substrates for TGase. No such change occurred in ovalbumin and deamidated gluten, applied as controls, under the influence of TGase. On the basis of our experiments it is postulated that the first step in gluten toxicity is presumably the binding of gluten to the intestine mucosa. In this binding the high transglutaminase activity in the intestines of coeliac patients and the high glutamine content of gluten may have an important role.

Celiac Disease↗

PubMed1987

Effect of the pKM101 plasmid on the repair of single-strand breaks in DNA induced by ionizing irradiation in Escherichia coli.

The effect of pKM101 plasmid on repair of single-strand breaks in DNA induced by 60Co-gamma irradiation in E. coli K12 AB1157 (wild type) and in its recA- and recB- mutant cells was studied by alkaline sucrose gradient sedimentation method. For quantitative analysis of sedimentation profiles we calculated the S1/2 values described by Veatch and Okada. The S1/2 values of unirradiated cells were 21.10, and after 200 Gray irradiation 11.35, due to the original incidence of single-strand breaks. The presence of pKM101 did not influence these values in either cases. This means that pKM101 had no effect on the rise of single-strand breaks in DNA. During a post-irradiation incubation period at 37 degrees C for 60 min the S1/2 value of the wild type strain increased from 11.35 to 19.22, that of the recB- from 11.50 to 15.23, while the S1/2 value of the recA- mutant did not change owing to the lack of repair of single-strand breaks. pKM101 plasmid markedly increased the S1/2 value in wild type strain and in recB- mutant, while it had no effect on S1/2 in recA- cells, during this post-irradiation incubation period. Thus the effect of pKM101 on the repair of single-strand breaks in DNA proved to be dependent on recA+ genotype. Nalidixic acid at 100 micrograms/ml concentration inhibited the repair of single-strand breaks in both wild type and recB- mutant cells harbouring pKM101 plasmid.

Centrifugation, Density Gradient↗

PubMed1987

Elimination of UV-resistance inducing effect of R46 R factor by 5-fluorouracil.

R-factor curing capacity of 5-fluorouracil (5-FU) was studied. Well detectable increase in UV-resistance was found in E. coli K12 AB1157 strain and its recB-, recC-, recF- mutants harbouring R46 R-factor. After treatment with 5-FU, these strains lost not only the antibiotic resistance coded for R46 R-factor but their UV-radioresistance, as well.

Dose-Response Relationship, Radiation↗

PubMed1986

Effect of a calcium-binding gluten fraction on the superprecipitation of actomyosin.

The Ca2+-binding gluten fraction isolated by the authors has been shown to prolong the clearing phase and to shorten the physiological contraction phase of ATP activated natural actomyosin suspension in the presence of physiological potassium chloride in a dose dependent manner. The mean ATPase activity of myosin and actomyosin was calculated for each phase from the actual ATP concentrations, measured at 30 sec intervals, and from the phase lengths. The preparation was found markedly to inhibit the ATPase activity during the clearing and physiological contraction phase. Since both myosin and actomyosin ATPase is Ca2+-dependent, it is assumed that the inhibitory effect of the gluten fraction on ATPase activity may be mediated by its free Ca2+-binding capacity, resulting in modified phase lengths of actomyosin superprecipitation. On the basis of these experimental results a hypothesis is put forward of the part of the Ca2+-binding gluten fraction played in the pathomechanism of coeliac disease.

Actomyosin↗

PubMed1985

Isolation and physicochemical and functional properties of a calcium binding gluten fraction.

Each milligram gluten protein isolated from bread contains 0.03-0.06 mumol calcium. On theoretical grounds we have concluded that this calcium quantity is bound to the free carboxyl groups not participating in peptide bonds of dicarbonic aminoacids, especially glutaminic acid, making up a large proportion within the aminoacids of gluten. After treatment with EGTA, a well-known calcium complex forming compound, two gluten fractions can be distinguished: water-soluble gluten-ES, and gluten-EP soluble in acetic acid. The aminoacid composition of gluten-ES is similar to that of unfractionated gluten. It is rich in aminodicarbonic acid (glu), aminodicarbonic acid amide (gln) and proline. Further properties of gluten-ES are: immunological similarity to gluten; a molecular mass of 36 000 dalton; an absorption maximum at 275.6 nm; a Ca2+-binding capacity of 0.72 mumol Ca2+/mg protein as measured by atomic absorption spectrophotometry and by Ca2+ ion selective electrode; inhibitory effect of a small quantity (25-30 micrograms) of the compound on the Ca2+-Mg2+ dependent ATPase and Ca2+-uptake of fragmented sarcoplasmatic reticulum. Preliminary experiments have demonstrated that gluten-ES has an influence on other calcium ion mediated systems like actomyosin superprecipitation. We put forward the hypothesis that by its Ca2+-binding capacity, gluten-ES is capable of influencing the level of free calcium and may thus play a part in the pathomechanism of coeliac disease.

Amino Acids↗

PubMed1985

Vesicle release from erythrocytes during storage and failure of rejuvenation to restore cell morphology.

During storage, discocyte-echinocyte transformation of erythrocytes occurs progressively and microvesicles release from the spicules in the form of membrane-encapsulated red cell fragments. After rejuvenation part of the membrane-deficient cells, mainly spheroechinocytes, transform into spherostomatocytes because of their inability of restoring the surface area to volume ratios of normal discocytes. The proportion of spherostomatocytes formed on rejuvenation increases with storage time. Numerical indices for characterizing the "level of reversion" and the "quality of reversion" were developed. Osmotic fragility curves and light scattering histograms also reflected a partially irreversible increase of inhomogeneity of stored cell populations.

2,3-Diphosphoglycerate↗