Biomedical subjects

J Kopecký

Publications and source records attributed to J Kopecký.

At least 19 recordsLinked to original sources

PubMed2000

Identification of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in Ixodes ricinus ticks from southern Bohemia using monoclonal antibodies.

The prevalence of Borrelia burgdorferi sensu lato (s.l.) in 761 Ixodes ricinus ticks collected in different localities of southern Bohemia was investigated. The minimum infection rate of nymphs, males and females as determined by dark-field microscopy was 4.8% in 1997. The total B. burgdorferi prevalence determined in ticks in 1998 was 11.04%. A significant local variability of the prevalence of B. burgdorferi was not found. Nine isolates were identified at a genospecies level by immunoblotting with prepared monoclonal antibodies (Mabs). The most prevalent genospecies in ticks was B. burgdorferi sensu stricto (s.s.) (6 strains). Two tick isolates reacted with Mabs specific to both B. garinii and B. afzelii and one isolate was detected as a mixed culture of B. burgdorferi s.s. and B. garinii. Considerable variability in protein composition among the strains obtained was found to exist. Five B. burgdorferi genospecies-specific Mabs were prepared and used for the identification of spirochaete isolates. Mabs 9B8 and 6C5 reacted with OspA and OspB proteins of B. burgdorferi s.s., while 4D7 and 1D11 recognised OspA protein of B. garinii and 9D2 specifically reacted with the OspB protein of B. afzelii. In addition, Mab 5H4 reacted with flagellin of all the above-mentioned genospecies of B. burgdorferi s.l.

Animals↗

PubMed1999

Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition.

Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN from S. cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions. Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E. coli and combined in vitro to reconstitute the active enzyme.

Acetolactate Synthase↗

PubMed1999

Transgenic UCP1 in white adipocytes modulates mitochondrial membrane potential.

To test if mitochondrial uncoupling in white adipocytes is responsible for obesity resistance of the aP2-Ucp transgenic mice expressing ectopic uncoupling protein 1 (UCPI) in white fat, mitochondrial membrane potential (delta psi(m)) was estimated by flow cytometry in adipocytes isolated from gonadal fat. Ectopic UCP1 (approximately 0.8 mol UCP1/mol respiratory chain) decreased the delta psi(m) and rendered the potential sensitive to GDP and fatty acids. These ligands of UCP1 had no effect on delta psi(m) in white adipocytes from non-transgenic mice, suggesting that the function of endogenous UCP2 in adipocytes was not affected. The results support the hypothesis that mitochondrial uncoupling in white fat may prevent development of obesity.

Adipocytes↗

PubMed1999

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle.

The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15-20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions.

Adaptation, Physiological↗

PubMed1999

Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-gamma by the upregulation of interleukin-10.

Tick saliva has been shown to modulate host immunity by a so far unknown mechanism. We have demonstrated an inverted effect of salivary gland extract (SGE), derived from partially fed Ixodes ricinus females, on the production of two cytokines, interferon (IFN)-gamma and interleukin (IL)-10, in vitro. While SGE markedly suppressed the elaboration of IFN-gamma by mouse splenocytes stimulated with lipopolysaccharide (LPS), the production of IL-10 was increased in comparison with SGE-untreated cultures. The suppressive effect of SGE could be abolished by the addition of an IL-10 neutralizing monoclonal antibody to splenocyte cultures. Similar results were obtained when live Borrelia afzelii spirochetes, which are transmitted in Europe by I. ricinus ticks, were used for the cytokine induction. These results suggest that tick saliva can upregulate the IL-10 production at the tick feeding site, which consecutively inhibits the elaboration of pro-inflammatory cytokines, for example IFN-gamma. This immunosuppression may facilitate the establishment of tick-transmitted pathogens in the host.

Animals↗

PubMed1999

A putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblotting.

Anti-idotypic monoclonal antibodies (anti-ID MAbs) were made against two mouse MAbs that neutralize the infectivity of the tick-borne encephalitis (TBE) virus. Three of the anti-ID MAbs (1) inhibited the binding of respective idiotypic MAb to the TBE virus antigen, (2) inhibited the infectivity of TBE virus when preincubated with virus-susceptible cells, and (3) bound to the surface of virus-susceptible but not virus-nonsusceptible cells. They recognized a 35-kD protein in immunoblotting analysis. Identification of this protein as a component of a putative TBE virus receptor was supported by the viroblot technique. In this assay, two polypeptide signals of 35 and 18 kD were obtained after incubation of blotted cell membrane proteins with the TBE virion antigen.

Animals↗

PubMed1999

Cytokine response to infection with the microsporidian, Encephalitozoon cuniculi.

The production of three cytokines, interferon gamma (IFN-gamma), interleukin 10 (IL-10) and interleukin 12 (IL-12), was measured after intraperitoneal infection of immunocompetent Balb/c mice and immunodeficient SCID mice with the microsporidian, Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923. High levels of IFN-gamma were detected in ex vivo cultures of peritoneal exudate cells (PEC) of Balb/c mice, a lower, but earlier IFN-gamma response was observed in PEC from SCID mice. The early IL-10 response was detected in ex vivo cultures of splenocytes from Balb/c but not from SCID mice, explaining a delay in the IFN-gamma response in Balb/c mice. IL-12 was detected in PEC cultures from SCID mice, indicating an alternative pathway of IFN-gamma production by NK cells stimulated by IL-12 derived from macrophages.

Animals↗

PubMed1998

High expression of uncoupling protein 2 in foetal liver.

To assess the putative role of mitochondrial uncoupling protein 2 (UCP2) during perinatal development, its expression was analysed in mice and rats. Expression was detected in a large range of foetal tissues. A unique developmental pattern of UCP2 expression was found in liver, where the level of UCP2 mRNA was about 30-fold higher in foetuses than in adults (mice data), and started to decline immediately after birth. Neither UCP1 nor UCP3 mRNA was expressed in foetal liver. As in adult liver, immunohistochemical analysis suggested exclusive localisation of UCP2 in the monocyte/macrophage cells. Our results indicate a role of UCP2 in haematopoietic system development.

Animals↗

PubMed1998

Acute effects of bezafibrate on blood pressure and renal haemodynamics in SHR and WKY rats.

BACKGROUND: Bezafibrate, a fibric acid analogue, has well-established lipid- and fibrinogen-lowering properties. Some data exist pointing towards a blood-pressure-lowering effect of bezafibrate. Thus the aim of this study was to examine the acute effect of bezafibrate on blood pressure and renal haemodynamics in hypertensive and normotensive rats. METHODS: 8 Wistar-Kyoto (WKY) and 12 spontaneously hypertensive rats (SHR) were treated with i.v. bolus injections of vehicle and 1-10 mg of bezafibrate in increasing doses every 15 min. Mean arterial pressure (MAP), renal blood flow (RBF), cortical blood flow (CBF), and medullary blood flow (MBF) were monitored continuously, together with plasma renin activity (PRA), urine volume and urinary Na+, K+, and protein concentration (15-min intervals). RESULTS: Bezafibrate reduced MAP in a dose-dependent manner (mean +/- SEM): in WKY, 1 mg bezafibrate, -1.13 +/- 0.61 mmHg and after 10 mg bezafibrate, -7.25 +/- 1.10 mmHg; in SHR, -0.60 +/- 0.43 and -5.83 +/- 0.90 mmHg respectively. In contrast to vehicle, bezafibrate induced a dose-dependent increase in RBF (WKY, 0.21 +/- 0.10 and 0.83 +/- 0.48 ml/min; SHR, 0.38 +/- 0.10 and 3.09 +/- 0.45 ml/min respectively) and a corresponding decrease in renal vascular resistance which was significantly greater in SHR than in WKY. The increase in RBF was paralleled by an increase in CBF. No effect of bezafibrate on MBF, PRA, urine flow, or urinary Na+, K+ or protein excretion was observed. The observed effects could not be attributed to one of the classic vasodilating mechanisms. CONCLUSIONS: We conclude that in rats bezafibrate is a potent hypotensive drug exhibiting additional effects on renal haemodynamics.

Animals↗

PubMed1998

Brown fat is essential for cold-induced thermogenesis but not for obesity resistance in aP2-Ucp mice.

The role of brown adipose tissue in total energy balance and cold-induced thermogenesis was studied. Mice expressing mitochondrial uncoupling protein 1 (UCP-1) from the fat-specific aP2 gene promoter (heterozygous and homozygous aP2-Ucp transgenic mice) and their nontransgenic C57BL6/J littermates were used. The transgenic animals are resistant to obesity induced by a high-fat diet, presumably due to ectopic synthesis of UCP-1 in white fat. These animals exhibited atrophy of brown adipose tissue, as indicated by smaller size of brown fat and reduction of its total UCP-1 and DNA contents. Norepinephrine-induced respiration (measured in pentobarbital sodium-anesthetized animals) was decreased proportionally to the dosage of the transgene, and the homozygous (but not heterozygous) transgenic mice exhibited a reduction in their capacity to maintain body temperature in the cold. Our results indicate that the role of brown fat in cold-induced thermogenesis cannot be substituted by increased energy expenditure in other tissues.

Adipose Tissue↗

PubMed1998

[Mitochondrial energy metabolism, uncoupling proteins and adipose tissue accumulation].

New possibilities are explored for supporting classical treatment of obesity. There are three main ways for the pharmacotherapy of obesity: (1) inhibition of absorption of nutrients in the intestine; (2) modulation of the activity of hypothalamic centra controlling satiety and food consumption; and (3) induction of energy dissipation in tissues (thermogenesis). The last possibility received more attention due to recent discoveries. The goal would be to affect mitochondrial energy conversion in a way leading to elevated thermogenesis and not to ATP production.

Adenosine Triphosphate↗

PubMed1998

Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro.

Tick saliva has been shown to contain a variety of pharmacologically active molecules, including those with immunosuppressive activities. There is increasing evidence that the nonspecific suppression of host immunity by tick saliva is exploited by tick-borne pathogens, e.g. the saliva-activated transmission (SAT) of some tick-borne viruses. We have demonstrated the suppressive effect of the salivary gland extract (SGE) derived from partially fed (five days) Ixodes ricinus females on important mechanisms of innate immunity: natural killer (NK) cells, interferon and nitric oxide production. SGE reduced the interferon induction by polyinosinic-polycytidylic acid (poly I:C) or Escherichia coli lipopolysaccharide (LPS) in a culture of Balb/c mouse splenocytes by 94% and 62%, respectively. SGE suppressed the cytotoxicity of nonstimulated and in vivo poly I:C-stimulated mouse NK cells by up to 31% and 26%, respectively. The induction of NK activity in vitro by LPS but not by Concanavalin-A (Con-A) was also downregulated in the presence of SGE. The addition of SGE to cultures of mouse macrophages partially inhibited the production of nitric oxide, induced by LPS. These data suggest that the facilitating effect of SGE on the transmission of some tick-borne pathogens might be associated with the suppression of the host innate resistance mechanisms, represented by interferon, nitric oxide and NK cells.

Animals↗

PubMed1998

Interaction of virulent and attenuated tick-borne encephalitis virus strains in ticks and a tick cell line.

An interference between a thermosensitive (ts) mutant and the wild-type (wt) of tick-borne encephalitis (TBE) virus in Ixodes ricinus L. and Rhipicephalus appendiculatus (Neumann) ticks is reported. I. ricinus females were dually infected by a parenteral inoculation of ts and wt strains at 10-day interval. Interference was demonstrated by the lowered ability of wt virus to replicate in ticks previously infected by ts virus. The wt virus was demonstrated in only 30% of the ticks; the average virus titre was lowered by 2.1 log10 compared with the control group, which was infected with the wt virus only. The oral infection of R. appendiculatus ticks with the same viruses also revealed an interference with the growth of the superinfecting wt virus. While in the control group all the ticks became infected, in the dually infected group the wt virus was found in only 50% of the ticks. However, when the ticks were infected orally with ts virus and superinfected parenterally with the wt virus, no interference was observed. In a R. appendiculatus-derived cell line persistently infected with the ts virus (100% of the cells), a partial inhibition of the growth of the superinfecting wt virus was observed. The ts virus retained its thermosensitive phenotype throughout the persistent infection of both the ticks and the tick cell line.

Animals↗

PubMed1997

Characterization of phytase produced by Aspergillus niger.

The extracellular activity of Aspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor. The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55 degrees C and high pH and temperature stability. The Km for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively. The enzyme is noncompetively inhibited by inorganic monophosphate (Ki = 2.85 mmol/L) and by Cu2+, Zn2+, Hg2+, Sn2+, Cd2+ ions and strongly by F- ones; it is activated by Ca2+, Mg2+ and Mn2+ ions. The substrate specificity of phytase is broad with the highest affinity to calcium phytate.

6-Phytase↗

PubMed1997

Tissue metabolism and plasma levels of thyroid hormones in critically ill very premature infants.

Thyroid status was characterized in very preterm infants (gestational age < or =32 wk; n = 61) from birth through d 14, and in infants who died within 16 d after delivery (n = 10), where it was also correlated with metabolism of iodothyronines in peripheral tissues (brain, liver, kidney, skeletal muscle, and adipose tissue). At 3 d of life, mean plasma levels of thyroxine, triiodothyronine, and TSH started to decrease, being lower in the critically ill compared with healthy premature neonates. Activities of the three iodothyronine deiodinases enzymes (type I, II, and III, respectively) were detected in all postmortem tissue samples, except for absence of the type II activity in kidney. All activities were the highest in liver and differed in other tissues. Lack of correlation between the type I activity in liver (and kidney), and plasma levels of thyroid hormones suggested that the thyroid was the primary source of circulating triiodothyronine. On the other hand, namely in brain, correlations between activity of the deiodinases and plasma hormone levels were found which suggested a complex control by thyroid hormones of their own metabolism. High activity of type III in liver, adipose tissue, and skeletal muscle demonstrated a role of these tissues in thyroid hormones degradation. Results support the view that peripheral tissues of very preterm infants are engaged in local generation of triiodothyronine, and inactivation of thyroid hormones, but do not represent a major source of circulating triiodothyronine.

Adipose Tissue↗

PubMed1997

[Trans-sphincteric resection of the rectum].

On the base of long time watching of 16 patients treated by transsphincteric resection of the rectum authors believe that this operation has even nowadays still its justification among operations preserving continention. They have dominant position namely in indications for benign disease, where only a small part of patients require a more extent performance. On the contrary, at rectum carcinoma the operating performance is highly elective.

Digestive System Surgical Procedures↗

PubMed1996

High-performance liquid chromatographic determination of tramadol in human plasma.

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.

Administration, Oral↗

PubMed1996

Possible use of excretion of tubular epithelial cells for the study of the nephrotoxic effect of xenobiotics.

The method of determination of the minute excretion of tubular epithelial cells renders it possible to investigate the course of the nephrotoxic effect of the toxin by the influence of which excretion of tubular round epithelial cells is increased. The nephrotoxic effect of repeated administration of amphotericin B (1 mg/kg, i.v.), which produced up to 12-fold increases in the number of excreted epithelial cells, was examined. Repeated administration of cyclosporin A (45 and 56 mg/kg, p.o.) produced up to 23-fold increases in the number of excreted epithelial cells. The degree of excretion of epithelial cells after administration of both drugs was compared with the urinary excretion of alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase, which indicated nephrotoxicity in amphotericin B and cyclosporin A with a lower sensitivity than the increase in the excretion of epithelial cells. In the experiment with cyclosporin A, urinary excretion of epithelial cells was further correlated with renal functional tests (clearance of polyfructosan and hippurate.

Acetylglucosaminidase↗