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Biomedical subjects

P Svoboda

Publications and source records attributed to P Svoboda.

At least 19 recordsLinked to original sources
PubMed1996

Cold-induced reduction in Gi alpha proteins in brown adipose tissue. Effects on the cellular hypersensitization to noradrenaline caused by pertussis-toxin treatment.

The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi 1 alpha and Gi 2 alpha, as well as of the beta-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (NA+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.

Acclimatization↗
PubMed1996

Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants.

Human embryonic kidney 293 cells that had been transfected to express the long isoform of the rat thyrotropin-releasing hormone (TRH) receptor (clone E2) were further transfected with a cDNA encoding the murine version of G11alpha. A clone was isolated (clone E2M11) that stably expressed murine as well as the endogenous human G11alpha. Subcellular fractionation demonstrated identical cellular distribution of the two species variants of this G protein. Sustained exposure of clone E2M11 cells to TRH resulted in substantial cellular redistribution and reduction in total cellular levels of G11alpha immunoreactivity. Fractions of both the exogenously introduced murine and endogenously expressed human isoforms of G11alpha were transferred from plasma membranes to low density membranes (detected as a shift from middle to low density regions on sucrose density gradients) and cytosol fractions. The plasma membrane redistribution to low density membrane was accompanied by a parallel redistribution of G protein beta subunits; however, there was no increase in beta subunits in the cytosol. The total cellular amount of G11alpha subunits was decreased to 21% and 59% for human and murine isoforms, respectively, and beta subunits were decreased to 68% after sustained treatment with TRH compared with controls (100%). Such data are consistent with the notion that the agonist-occupied long isoform of the rat TRH receptor may be able to partially differentiate between the endogenous (human) and exogenous (murine) G11alpha. This was not a reflection that the murine G protein was expressed but incorrectly folded as both species variants of G11alpha were solubilized equally from E2M11 membranes by sodium cholate. Using this system, we demonstrate both agonist-induced subcellular redistribution and down-regulation of G11alpha and beta subunit proteins in response to activation of a phospholipase C coupled receptor.

Amino Acid Sequence↗
PubMed1996

[Stress ulcers in patients with polytrauma].

Authors treat in their institution yearly 100-120 patients with polytrauma. In the Endoscopic Centre of the hospital approx. 20 gastroduodenal ulcers are diagnosed yearly in patients with supposed stress etiology. Authors analyze the pathophysiology of origin of stress ulcer, clinical symptoms and acute diagnostics. They evaluate results of early surgical treatment. Authors place the great importance on explicit treatment methods for prevention of stress ulcer origin.

Humans↗
PubMed1995

[Cytokine levels in patients with multiple injuries].

BACKGROUND: The role of cytokines in trauma still has not been satisfactorily elucidated. Multiorgan failure (MOF) development should be also under the direction of cytokines, endotoxin, and other mediators. METHODS AND RESULTS: Therefore we prospectively studied 88 patients with multiple trauma admitted to Traumatological Hospital Brno from June 1, 1992 to May 31, 1993. Extent of the trauma was determined by Injury Severity Score (ISS), Revised Trauma Score (RTS), and TRISS methodology with probability of survival. In study patients was investigated concentrations of interleukin 1, 2, 6, and tumor necrosis factor (TNF). Of above mentioned cytokines were elevated only IL-6 levels at admission and significant correlation with ISS was found (r = 0.32; p < 0.01). MOF developed in 23 patients (12 of them died), but it was not possible to predict the MOF development nor surviving according to admission levels of cytokines. A significant difference was observed in IL-6 levels of MOF patients one day before death (439 +/- 111 ng/l) in comparison with MOF patients, who survived (132 +/- 88 ng/l, p < 0.001). None of 12 MOF patients with IL-6 concentrations above 400 ng/l survived. CONCLUSIONS: We conclude that IL-6, less TNF, seems to play an important role in organism response to multiple trauma, and later elevation in these cytokines levels, especially IL-6 level, in MOF patients mean poor prognosis. We had found a significant correlation between initial IL-6 level and ISS. Other cytokines did not show changes during the study.

Adolescent↗
PubMed1995

Purification and characterization of three alpha 2-antiplasmin and alpha 2-macroglobulin inactivating enzymes from the venom of the Mexican west coast rattlesnake (Crotalus basiliscus).

Three distinct alpha 2PI (alpha 2-antiplasmin) degrading and alpha 2M (alpha 2-macroglobulin) inhibiting enzymes, named proteinase a, b and c, have been purified from the venom of Crotalus basiliscus (the Mexican west coast rattlesnake) by fast protein liquid chromatography (anion-exchange chromatography and gel filtration chromatography). SDS-PAGE revealed that proteinase a and b had similar mol. wts (approximately 23,500), whereas proteinase c displayed a mol.wt of approximately 24,200. Their isoelectric points were found to be acidic, ranging from pH 4.8 to 5.7. The proteinase activity of all three enzymes was inhibited in the presence of EDTA. Dependent on enzyme concentration, a progressive and catalytic inactivation of alpha 2PI was induced, leading to an almost complete loss of the plasmin inhibitory activity at a molar ratio of enzyme: alpha 2PI = 0.1 within 60 min. The ability of alpha 2M to protect the esterolytic activity of trypsin from inhibition by soybean trypsin inhibitor was only reduced at a molar ratio of enzyme: alpha 2M = 0.5, whereas no inactivation could be observed when the three venom proteinases were incubated with an excess of alpha 2M, suggesting that the inactivation occurred by complex formation but not by degradation of the intact alpha 2M molecule. In SDS-PAGE, inactivation of human alpha 2PI (mol. wt 68,000) correlated with the appearance of two cleavage products with an approximate mol. wt of 56,000 and 11,000, respectively. The three proteinases had no thrombin-like activity. Plasminogen and factor X were not activated and no platelet aggregation was induced. They degraded the A alpha- and B beta-chain of fibrinogen and showed plasma extravasation-inducing activity following intradermal injection into the abdominal skin. None of the enzymes showed any activity against a series of chromogenic p-nitroanilide substrates.

Animals↗
PubMed1994

The short and long forms of the alpha subunit of the stimulatory guanine-nucleotide-binding protein are unequally redistributed during (-)-isoproterenol-mediated desensitization of intact S49 lymphoma cells.

We report here that desensitization of the beta-adrenergic receptor-triggered transmembrane signalling in S49 wild-type lymphoma cells, induced by (-)-isoproterenol (1 microM), results in unequal intracellular redistribution of the splicing variants of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory (Gs alpha) protein (Gs alpha-short and Gs alpha-long) and alters the functional characteristics of the membrane-associated signal transduction complex. We found that two cellular pools of membranes, light-density membranes and plasma membranes prepared by sucrose-density-gradient centrifugation of cell homogenates differed in their content of Gs alpha splicing subforms and, moreover, that prolonged activation of the beta-adrenergic pathway induced intermembrane redistribution of the splicing variants of Gs alpha. Short (10 min) as well as prolonged (1 h) (-)-isoproterenol treatment of the cells shifted Gs alpha-short from light-density membranes to plasma membranes and increased the total amount of light-density membrane-bound Gs alpha-long; in parallel, the maximal (-)-isoproterenol-stimulated or AlF4(-)-stimulated adenylyl cyclase activities measured in the plasma membrane pools prepared from treated cells decreased. The functional characteristics of the membrane-bound Gs alpha pools were examined by a cyc(-)-reconstitutive adenylyl cyclase assay where extracts of the plasma membrane and light-density-membrane pools, respectively, were mixed with plasma membranes derived from the mutant S49 cell line, cyc-, lacking Gs alpha. The maximal cyc(-)-reconstitutive activities of the extracts prepared from light-density membranes of short-term as well as long-term desensitized cells increased compared to control cells. These findings may indicate differences in the functioning of the splicing variants of Gs alpha.

Adenylyl Cyclases↗
PubMed1994

Agonist-induced transfer of the alpha subunits of the guanine-nucleotide-binding regulatory proteins Gq and G11 and of muscarinic m1 acetylcholine receptors from plasma membranes to a light-vesicular membrane fraction.

A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist-specific down-regulation of both Hm1 receptors and the alpha subunits of the guanine-nucleotide-binding (G)-proteins Gq and G11 which is accompanied by a desensitization of inositol-phospholipid-specific-phospholipase-C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125-131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gq alpha and G11 alpha and of Hm1 receptors was assessed after short-term and long-term treatment with carbachol. Short-term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both Gq alpha and G11 alpha and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gq alpha and G11 alpha in each cell remained unchanged under these conditions. A similar transfer was noted for the G-protein Gs alpha but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gs alpha subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gq alpha and G11 alpha proteins were down-regulated to 25% and 40%, respectively, when compared with untreated cells. Such observations support the idea that down-regulation of both the Hm1 receptor and its associated inositol-phospholipid-specific-phospholipase-C-linked G-proteins is produced by two sequential steps. The first is a transfer of signal-transducing polypeptides from the plasma membrane to a non-plasma membrane light vesicle fraction. The second step is represented by an agonist-specific down-regulation pathway. Both the Hm1 receptor and Gq alpha/G11 alpha appear to follow similar sequestration and down-regulation patterns.

Adenosine Triphosphatases↗
PubMed1994

Dynamics of interleukin 1, 2, and 6 and tumor necrosis factor alpha in multiple trauma patients.

The involvement of cytokines in trauma still has not been satisfactorily elucidated. The development of multiorgan failure, the very serious complication of multiple trauma with high mortality, should also be controlled by cytokines, endotoxin, and other mediators. We therefore prospectively studied 42 consecutive patients with multiple trauma admitted from June to December 1992 to the Research Institute for Traumatology and Surgery in Brno. Study patients were characterized by Injury Severity Score (ISS), Revised Trauma Score, and TRISS methodology. In all patients, tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 1, 2, and 6 levels were investigated. Of the cytokines, only IL-6 levels were elevated at admission and significant correlation with ISS was observed (r = 0.735; p < 0.001). Multiple organ failure (MOF) developed in 14 patients (seven died) and it was not possible to predict this MOF development nor survival by initial cytokine levels. A significant difference was observed when IL-6 concentrations one day before death (423 +/- 105 pg/mL) were compared with the highest concentrations in MOF survivors (112 +/- 71 pg/mL; p < 0.001). This difference was found also for TNF (528 +/- 314 pg/mL vs. 216 +/- 165 pg/mL; p < 0.05). None of six MOF patients with IL-6 > 400 pg/mL survived. In conclusion, the IL-6 and TNF-alpha levels seem to play a significant role in multiple trauma and their late elevation in patients with MOF conveyed a poor prognosis. A significant correlation between initial IL-6 levels and ISS was observed. Other cytokines did not show dynamic changes during the study.

Adult↗
PubMed1994

[Serum levels of cytokines as a measure of response to stress after various types of elective gallbladder surgery].

The authors investigated the cytokine levels in patients after laparoscopic cholecystectomy (LCHE), conventional open cholecystectomy (OCHE) and complicated open cholecystectomy (KOMPL) in order to assess whether there is a relationship between cytokine levels and the general reaction of the organism, the type and extent of the operation. They did not find an increase of IL-1 or IL-2 levels in any of the patient groups. The IL-6 concentration was slightly raised only in three patients of the OCHE group three hours after surgery and this rise persisted for 24 hours. In the KOMPL group there was a more marked rise of IL-6 in 9 patients within 3 hours after surgery, persisting in 6 patients for 24 hours and in 2 patients for 48 hours. The TNF values were similar; in group OCHE they were slightly elevated in 2 patients 24 hours after operation. In the KOMPL group these values were elevated in 5 patients for 3 hours after surgery and this increase persisted in 2 for 24 hours after surgery. Based on the investigation of cytokine levels, which is a recent indicator for evaluating the reaction of the organism to stress, conclude that laparoscopic cholecystectomy is a minimal stress for the patient and is associated with a zero defence reaction of the organism, if evaluated according to serum cytokine concentrations.

Adult↗
PubMed1994

[Hemopurification methods in the surgical unit].

The authors submit their initial experience with the activities of the hemodialyzation centre at a surgical department. They present an analysis of 79 patients where some hemopurifying procedures or their combinations were applied. These methods make it possible to perform more safely extensive surgery, they improve the care of patients with multiple injuries and extend therapeutic possibilities in acute pancreatitis and hyperbilirubinaemia when they cannot be treated by other methods. From the range of hemopurifying methods they consider the following most suitable for a surgical department: classical acute haemodialysis, hemodiafiltration, haemoperfusion and continuous arteriovenous dialysis.

Acute Kidney Injury↗
PubMed1994

[Is it possible to predict a decrease in portal pressure after administration of ACE inhibitors?].

We have previously shown that angiotensin converting enzyme inhibitor enalapril causes a potent decrease in portal pressure gradient, but only in about one half of patients with portal hypertension and an episode of bleeding esophageal varices in patient's history. Twenty-one consecutive patients after first episode of bleeding from esophageal varices were enrolled in the trial. Patients were treated by sclerotherapy in combination with enalapril. The level of ACE in patients with portal hypertension (10.4, SD 4.5) was significantly higher than in normal population (4.5, SD 1.3 mu kat/l-1) [p < 0.001]. After 3 months treatment decreased ACE to normal or subnormal levels in all 21 patients (2.9, SD 1.5 mu kat.l-1) [p < 0.001], but simultaneously measured hepatic venous pressure gradient decreased more than 3 mm Hg only in 11 (52%). No correlation between changes of portal pressure gradient and changes of ACE concentrations were found. We conclude that patients with portal hypertension have significantly higher serum ACE level with a large decrease after enalapril, but it is not possible to predict the effect of enalapril on portal pressure by estimation of ACE level in serum in individual patient.

Adolescent↗
PubMed1993

Attenuation of Gs alpha coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters.

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5'-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.

Acclimatization↗
PubMed1993

[Another source of hemorrhage--risks for patients with esophageal varices].

The authors treated during the past three years 312 patients with oesophageal varices after the first haemorrhage. All patients were treated by endoscopic sclerotization of oesophageal varices and drugs which reduce the excessive portal pressure. After a minimum of two sclerotherapeutic sessions, following control of acute haemorrhage, the authors observed a relapse of haemorrhage from the upper gastrointestinal tract in 38 patients. In 20 of them the relapse of haemorrhage was again from oesophageal varices, but in 18 patients it was of different origin and would not be affected by classical treatment with a Sengstaken tube. The authors draw attention to the necessity of emergency endoscopy in these patients and to the fact that possible postponement of rational treatment, e.g. in duodenal ulcers insertion of a tube, may threaten the patient's life.

Adolescent↗
PubMed1992

Plasma-membrane-independent pool of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein in a low-density-membrane fraction of S49 lymphoma cells.

We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the alpha subunit of Gs (Gs alpha) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900-7903], three membrane-bound forms of Gs alpha were identified through rate-zonal centrifugation in sucrose density gradients, Gs alpha-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gs alpha in the high-density portion of the gradient (1.13-1.16 g/cm3) is identical with that of beta-adrenergic-receptor binding, Na/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09-1.11 g/cm3), contains a much lower total amount of Gs alpha and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH-cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of Gs alpha located at the top of the gradient (1.06-1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of Gs alpha is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100,000 x g proves its particulate (membrane) character. The light membrane form of Gs alpha subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of Gs alpha. Overall, our data indicates that a substantial portion of Gs alpha is localized in membrane pools other than plasma membrane.

Adenylyl Cyclases↗
PubMed1992

[How should treatment of esophageal varices proceed?].

The authors submit their experience and data from the literature on the problem of oesophageal varices. In haemorrhage of varices at present the most successful procedure is endoscopic haemostasis concurrently with intensive treatment focused on the basic disease and replacement of blood losses. In patients with a history of haemorrhage from varices endoscopic sclerotization is generally recommended. The authors supplement it with the promising medicamentous reduction of the portal pressure. Other procedures, i.e. surgery, are indicated only in a minority of patients whose varices do not reposed to haemostasis and medicamentous reduction of high portal pressure. The problem how to proceed in varices which did not bleed so far is still unresolved. The authors recommend individual evaluation and submit their own procedure.

Esophageal and Gastric Varices↗