PubMed Health⌕ Search

Biomedical subjects

S Fischer

Publications and source records attributed to S Fischer.

At least 19 recordsLinked to original sources
PubMed1992

Spleen protein tyrosine kinases TPK-IIB and CSK display different immunoreactivity and opposite specificities toward c-src-derived peptides.

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48-64 and 353-369 sequences of CSK, a protein tyrosine kinase implicated in the down-regulation of src-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase present in spleen but they do not cross-react with purified TPK-IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin-Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp60c-src, namely Tyr-416 and Tyr-527. These data support the view that TPK-IIB and CSK may exert opposite effects on the activity of src-related protein tyrosine kinases.

Amino Acid Sequence↗
PubMed1992

Binding of recombinant variants of human tissue-type plasminogen activator (t-PA) to human umbilical vein endothelial cells.

Endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-PA) which is thought to be the major determinant of fibrinolytic activity in the blood. Most recently, a receptor protein for t-PA on human umbilical vein endothelial cells (HUVEC) in culture has been described (1); there are, however, in addition low affinity binding sites for t-PA on HUVEC. The sites of binding are of particular interest, because they are potential regulators of t-PA activity and clearance. We analysed the low affinity binding of recombinant t-PA (rt-PA) to normal diploid HUVEC and to the permanent human cell lines Jurkat, Daudi, HL 60 and K562 by flow cytometry applying t-PA specific monoclonal antibodies. Using this test system binding of both recombinant glycosylated human t-PA produced in Chinese hamster ovary cells (CHO-t-PA) and of nonglycosylated t-PA, produced in E. coli (BM 06.021) was investigated. Analysis of the binding pattern to HUVEC and other cell lines revealed that deglycosylation of full length rt-PA increases non-specific binding. Additionally, we investigated the binding properties of an unglycosylated t-PA deletion variant which comprises the kringle 2 and the protease domains (BM 06.022). Data obtained show that deletion of these domains most drastically reduces non-specific binding to HUVEC and other human cell lines.

Antibodies, Monoclonal↗
PubMed1992

Schizophrenic patients treated with high dose phenothiazine or thioxanthene become deficient in polyunsaturated fatty acids in their thrombocytes.

Total fatty acids were analysed in thrombocytes of schizophrenic patients treated with a "high dose" or "low dose" monotherapy of neuroleptic drugs phenothiazine or thioxanthene. The ratio of the very long chain fatty acid hexacosanoic acid to the long chain fatty acid docosanoic acid (C26:0/C22:0) increased in the "high dose" and "low dose" groups as compared to healthy untreated controls (P less than 0.05). The polyunsaturated fatty acid arachidonic acid decreased in the "high" and "low dose" groups (P less than 0.01 and P less than 0.05). The polyunsaturated fatty acids alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were not detectable in most of the "high dose" schizophrenic patients, however, they were found in the "low dose" group and in the controls. There was a negative correlation between the daily dosage of phenothiazine and the percentages of the polyunsaturated fatty acids arachidonic acid and alpha-linolenic acid+eicosapentaenoic acid+docosahexaenoic acid in thrombocytes (r = -0.87, P less than 0.01 and r = -0.81, P less than 0.01). Two patients of the "high dose" group with an especially high and long lasting monotherapy of neuroleptics were nearly devoid of polyunsaturated fatty acids in their thrombocytes. Untreated schizophrenic patients exhibited a fatty acid pattern in their thrombocytes not markedly different from that of the healthy untreated control group. We conclude that neuroleptic drugs phenothiazine or thioxanthene can alter the fatty acid pattern of thrombocytes.

Adult↗
PubMed1992

Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors.

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.

Blood Platelets↗
PubMed1992

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells.

Using the human erythropoietin-responsive hematopoietic cell line UT-7, we showed that erythropoietin (Epo) rapidly and specifically induced the tyrosine phosphorylation of its own receptor (M(r) 75,000) and increased the tyrosine phosphorylation of other proteins of M(r) 140,000, 120,000, 95,000, 60,000, 57,000, and 42,000. Neither granulocyte-macrophage colony-stimulating factor, interleukin 3, interleukin 6, nor the kit ligand induced the phosphorylation of the M(r) 75,000 receptor protein, although these growth factors induced the phosphorylation of other proteins. Cross-linking experiments using 125I-Epo indicated that the UT-7 cells expressed three Epo receptor subunits, of M(r) 100,000, 85,000, and 75,000, among which only the M(r) 75,000 subunit was tyrosine-phosphorylated following activation with Epo.

Blotting, Western↗
PubMed1992

[Acquired hemoglobin H disease in the early stage of erythroleukemia].

At the onset of erythroleukemia, the patient, a 74-year-old Swiss male, was also found to have microcytic-hypochromic anemia (Hb: 82 g/l, MCV: 69 fl, MCH: 21 pg). Further laboratory examinations revealed reduced hemoglobin stability, a hemoglobin H fraction of 3.0% on cellulose acetate-electrophoresis, and an abundance of hemoglobin H inclusion bodies in red cells. These findings, as well as the Swiss origin of the patient and his age at the onset of the disease, were consistent with acquired hemoglobin H disease. In addition to genetic hemoglobin H disease, acquired hemoglobin H disease was reported to be associated with myelodysplastic and myeloproliferative syndrome, or erythroleukemia and acute myelogenous leukemia. The literature contains fewer than 50 cases. It is suggested that the molecular basis of this rare disease involves a gene in trans to the alpha-globin genes reducing the expression of all four alpha-globin genes to approximately 10% of normal activity.

Aged↗
PubMed1992

Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.

Antibodies, Monoclonal↗
PubMed1992

Effect of ursodeoxycholic acid on the kinetics of the major hydrophobic bile acids in health and in chronic cholestatic liver disease.

Beneficial effects of ursodeoxycholic acid in chronic cholestatic liver diseases have been attributed to displacement of hydrophobic bile acids from the endogenous bile acid pool. To test this hypothesis, we determined pool sizes, fractional turnover rates, synthesis/input rates and serum levels of deoxycholic acid and chenodeoxycholic acid before and 1 mo after the start of treatment with ursodeoxycholic acid (13 to 15 mg/kg body wt/day) in four healthy volunteers and five patients with chronic cholestatic liver diseases (three with primary biliary cirrhosis and two with primary sclerosing cholangitis). Bile acid kinetics were determined by combined capillary gas chromatography-isotope ratio mass spectrometry in serum samples after administration of [2H4] deoxycholic acid and [13C]chenodeoxycholic acid. In healthy volunteers, deoxycholic acid pool sizes decreased during administration of ursodeoxycholic acid by 72%. In patients with cholestatic liver diseases, deoxycholic acid pool sizes before ursodeoxycholic acid treatment were only 13% of those in healthy volunteers and were unaffected by ursodeoxycholic acid treatment. Chenodeoxycholic acid pool sizes were not different in healthy volunteers and in patients with cholestatic liver disease, and were not altered by ursodeoxycholic acid treatment. In both healthy volunteers and patients with cholestatic liver disease, synthesis/input rates and serum levels of deoxycholic acid and chenodeoxycholic acid were not altered by ursodeoxycholic acid treatment. Because in our patients improvement of serum liver tests during short-term ursodeoxycholic acid treatment was noted without a decrease of the pool sizes of the major hydrophobic bile acids, we conclude that displacement of hydrophobic endogenous bile acids is not the mechanism of action of ursodeoxycholic acid in chronic cholestatic liver disease.

Adult↗
PubMed1992

Dietary N-3 polyunsaturated fatty acids decrease biliary cholesterol saturation in gallstone disease.

Because fatty acid composition of biliary phospholipids influences cholesterol secretion into bile, we investigated whether replacement of n-1 monounsaturated or n-6 polyunsaturated fatty acids with n-3 polyunsaturated fatty acids in biliary phosphatidylcholines reduces supersaturation with cholesterol and prevents precipitation of cholesterol crystals in bile of gallstone patients. Seven patients with radiolucent gallstones in functioning gallbladders were studied before (control) and after 5 wk of dietary supplementation with marine fish oil (11.3 gm/day = 3.75 gm n-3 polyunsaturated fatty acids/day). Duodenal bile was collected for analysis during intravenous infusion of cholecystokinin. Gallbladder emptying in response to cholecystokinin was comparable before and during intake of n-3 polyunsaturated fatty acids. Intake of n-3 polyunsaturated fatty acids increased (p less than 0.001) the fractions of eicosapentaenoic and docosahexaenoic acids and decreased the fractions of linoleic (p less than 0.001) and arachidonic acids (p less than 0.02) in biliary phospholipids. Concomitantly, the molar ratio of cholesterol to phospholipids decreased (-19%; p less than 0.05). As a consequence, the cholesterol saturation index was reduced by -25% (p = 0.01), from 1.60 +/- 0.44 to 1.24 +/- 0.38. However, in vitro nucleation time of duodenal bile was not prolonged. The decrease in cholesterol saturation was not sufficient to prevent nucleation of cholesterol crystals in bile of gallstone patients. In conclusion, our data suggest that cholesterol saturation can be influenced by the fatty acid composition of the phosphatidylcholines secreted in bile.

Adult↗
PubMed1992

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

Amino Acid Sequence↗
PubMed1992

Pharmacokinetic and thrombolytic properties of unglycosylated recombinant tissue-type plasminogen activator (BM 06.021) produced in Escherichia coli.

Recombinant tissue-type plasminogen activator (rt-PA) was produced in Escherichia coli cells in order to obtain an unglycosylated rt-PA (BM 06.021) with increased thrombolytic potency due to altered pharmacokinetic properties. The pharmacokinetics were studied in rabbits upon intravenous infusion of 200 kU/kg over 30 min. The thrombolytic dose-response effects were evaluated in a rabbit model with 125I-labeled venous thrombi upon intravenous infusion over 4 h. The thrombolytic effects after intravenous bolus injection of 200 kU/kg BM 06.021 were investigated in a canine model of coronary artery thrombosis. All studies were performed comparing BM 06.021 with glycosylated rt-PA (alteplase). BM 06.021 demonstrated a longer (p less than 0.05) half-life (5.6 +/- 2.6 vs. 2.1 +/- 0.3 min) and a lower (p less than 0.05) clearance rate (7.5 +/- 0.8 vs. 22.2 +/- 3.1 ml.min-1.kg-1) than alteplase in rabbits upon intravenous infusion. The dose-response curve of BM 06.021 for thrombolysis in a rabbit model of jugular vein thrombosis was located to the left of that for alteplase with a 2.1-fold lower effective dose of 50% thrombolysis (ED50) of BM 06.021 (207 vs. 436 kU/kg). Intravenous bolus injection of 200 kU/kg BM 06.021 induced the same reperfusion rate (4/6) as intravenous infusion of 800 kU/kg alteplase over 90 min in a canine model of coronary artery thrombosis. The residual thrombus wet weight did not significantly differ between BM 06.021 and alteplase (5.7 +/- 1.8 vs. 6.3 +/- 1.1 mg). The results indicate that unglycosylated rt-PA (BM 06.021) has a higher in vivo thrombolytic potency than glycosylated rt-PA (alteplase).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Training-overtraining: influence of a defined increase in training volume vs training intensity on performance, catecholamines and some metabolic parameters in experienced middle- and long-distance runners.

The influence of an increase in training volume (ITV; February 1989) vs intensity (ITI; February 1990) on performance, catecholamines, energy metabolism and serum lipids was examined in two studies on eight, and nine experienced middle- or long-distance runners; seven participated in both studies. During ITV, mean training volume was doubled from 85.9 km.week-1 (pretrial phase) to 174.6 km within 3 weeks. Some 96%-98% of the training was performed at 67 (SD 8)% of maximal performance. During ITI, speed-endurance, high-speed and interval runs increased within 3 weeks from 9 km.week-1 (pretrial phase) to 22.7 km.week-1 and the total training distance from 61.6 to 84.7 km.week-1. The ITV resulted in stagnation of running velocity at 4 mmol lactate concentration and a decrease in total running distance in the increment test. Heart rate, energy metabolic parameters, nocturnal urinary catecholamine excretion, low density, very low density lipoprotein-cholesterol and triglyceride concentrations decreased significantly; the exercise-related catecholamine plasma concentrations increased at an identical exercise intensity. The ITI produced an improvement in running velocity at 4 mmol lactate concentration and in total running distance in the increment test; heart rate, energy metabolic parameters, nocturnal catecholamine excretion, and serum lipids remained nearly constant, and the exercise-related plasma catecholamine concentrations decreased at an identical exercise intensity. The ITV-related changes in metabolism and catecholamines may have indicated an exhaustion syndrome in the majority of the athletes examined but this hypothesis has to be proven by future experimental studies.

Adult↗
PubMed1992

Inexpensive population screening for abnormal cells from the uterine cervix and valid results using the vaginal cytopipette.

Women who have not accepted an invitation by their own general practitioner to have a smear test, or for whom voluntary population screening projects are unavailable because of lack of public funds, may be at increased risk of malignant cervical changes. Such women may be offered self-testing by a cytopipette on the basis of the present analysis of abnormal cell counts and cell density on the cytological slides. As one link in a prophylactic study using the cytopipette, 107 women with abnormal cell findings were reinvited for simultaneous sampling by irrigation smear and by ordinary technique. The cytological findings indicate that the two methods are equal in recovery of abnormal epithelium, provided that the scanned quantity of cells is increased fourfold in the irrigation smear method, this quantity being distributed over two specimens each with double cell density and giving at least 1800 squamous epithelial cells per visual field (125:1) or a total of approximately 10(6) epithelial cells in each cytological specimen. Under these conditions the two sampling methods have the same predictive value.

Cell Count↗
PubMed1992

Interleukin 4 inhibits IL-2-induced proliferation of a human T-leukemia cell line without interfering with p56-LCK kinase activation.

Recently we described the establishment in culture and the immunophenotypic and functional characteristics of a human T-leukemia line TALL-103/2 derived from the T-cell receptor (TCR)-gamma/delta subset of T-lymphocytes. TALL-103/2 cells are absolutely dependent on interleukin 2 (IL-2) for their growth and survival in culture and thus provide a model cell line for studies of IL-2 signal transduction in a TCR-gamma/delta T-cell. In this report, we focus on the regulation of SRC-family protein tyrosine kinases (PTKs) by IL-2. TALL-103/2 cells were found to contain p56-LCK, p59-FYN, p62-YES and p53/56-LYN. Stimulation of growth factor-deprived TALL-103/2 cells with IL-2, however, induced increases in the relative activity only of the p56-LCK kinase. This IL-2-mediated increase in LCK kinase activity was manifested both by increased kinase autophosphorylation and by increased phosphorylation of the exogenous substrate enolase during in vitro kinase assays. Furthermore, immunoblot assays determined that the levels of p56-LCK protein were unaltered by IL-2-treatment, indicating that the measured elevations in LCK kinase activity reflected an increase in the specific activity of this PTK. In TALL-103/2 cells, IL-2 stimulated concentration-dependent increases in p56-LCK activity that displayed rapid and transient kinetics: detectable increases occurred within 1 minute after IL-2 stimulation, peaked at 10 minutes, and declined to baseline levels by 30 minutes. Treatment of TALL-103/2 cells with IL-4 abrogated IL-2-initiated proliferation, but did not inhibit IL-2-mediated activation of p56-LCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-Response Relationship, Drug↗
PubMed1992

Interaction of human immunodeficiency virus glycoprotein 160 with CD4 in Jurkat cells increases p56lck autophosphorylation and kinase activity.

The tyrosine protein kinase p56lck, specifically expressed in lymphoid cells, undergoes modifications of its autophosphorylation and kinase activity when these cells are triggered by mAbs to the T cell determinants. The kinase activity and the autophosphorylation of p56lck were analysed following triggering Jurkat cells with the human immunodeficiency virus (HIV) glycoprotein gp160 which interacts with CD4: both the autophosphorylation and the kinase activity are increased within 1-5 min following addition of gp160, this increase is maximum at 5 min and is followed by a gradual return to the basal level within 2 h. Similar to observations made with anti-CD4 mAbs the increase in kinase activity of p56lck is not associated with changes in the gel mobility nor is it associated with T cell activation. Triggering of T cells with a combination of anti-CD3 mAbs which activate T cells but not p56lck and gp160 greatly potentiated the increase of p56lck autophosphorylation and kinase activity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Training-overtraining: performance, and hormone levels, after a defined increase in training volume versus intensity in experienced middle- and long-distance runners.

Performance and hormones were determined in eight middle- and nine long-distance runners after an increase in training volume (ITV, February 1989) or intensity (ITI, February 1990). Seven runners participated in both studies. The objective was to cause an overtraining syndrome. The mean training volume of 85.9 km week-1 increased within 3 weeks to 176.6 km week-1 during ITV and 96-98% of training volume was performed as long-distance runs at mean(s.d.) 67(8)% of maximum capacity. Speed endurance, high-speed and interval runs averaging 9 km week-1 increased within 3 weeks to 22.7 km during ITI, and the total volume increased from 61.6 to 84.7 km. A plateau in endurance performance and decrease in maximum performance occurred during ITV, probably due to overtraining, with performance incompetence over months. Nocturnal catecholamine excretion decreased markedly (47-53%), contrary to exercise-related plasma catecholamine responses, which increased. Resting and exercise-related cortisol and aldosterone levels decreased. Improvement in endurance and maximum performance occurred during ITI indicating a failure to cause an overtraining syndrome in ITI. Decrease in noctural catecholamine excretion was clearly lower (9-26%), exercise-related catecholamine responses showed a significant decrease, cortisol and aldosterone levels remained almost constant, exercise-related prolactin levels decreased slightly. There were no differences in insulin, C-peptide, free testosterone, somatotropic hormone (STH), follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4). The decrease in nocturnal catecholamine excretion during ITV might indicate a decrease in intrinsic sympathetic activity in exhausted sportsmen. But it remains open whether this reflected a central nervous system incompetence.

Adult↗