Biomedical subjects

G E Staal

Publications and source records attributed to G E Staal.

At least 55 records · Page 3Linked to original sources

PubMed1989

Phosphorylation of 6-phosphofructokinase in rat lung.

1. 6-Phosphofructokinase of both fetal and adult rat lung consists of L, M and C subunits in a ratio of 65:25:10. 2. 6-Phosphofructokinase was purified to homogeneity from adult rat lung and subjected to phosphorylation in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. 3. This resulted in phosphorylation of the L and M subunit of 6-phosphofructokinase. 4. The C subunit was not phosphorylated. 5. However, if the phosphorylation of 6-phosphofructokinase was studied in the cytosol fraction of either fetal or adult lung using endogenous protein kinase(s), only the L subunit was phosphorylated. 6. This phosphorylation was dependent on cyclic AMP. 7. No influence of calcium, calmodulin or phosphatidylserine/diolein on the phosphorylation was observed. 8. It is concluded that although both L and M subunits of rat lung 6-phosphofructokinase are potential substrates for cyclic AMP-dependent protein kinase, their phosphorylation in situ is differentially regulated.

Aging↗

PubMed1989

Tyrosine protein kinase activity in normal and leukaemic human blood cells.

Tyrosine protein kinase (TPK) activity was measured in subcellular fractions of normal granulocytes, lymphocytes and monocytes, and acute and chronic myeloid and lymphoid leukaemic cells. Of several tested tyrosine-containing substrates, poly (glutamic acid: tyrosine = 4:1) (S1) proved to be the best synthetic substrate. High cytosolic TPK activity was found in every cell type. Different TPKs may exist in various cell fractions, as was indicated by the difference in Km values for S1 in cell fractions of normal granulocytes and lymphocytes. No significant difference was found in total TPK activity between normal and leukaemic cells, indicating that total TPK activity is not related to the leukaemic process itself. A highly significant difference was found in membrane fractions in normal granulocytes and M1-M2 AML cells versus normal monocytes and M4-M5 AML cells, suggesting an association between TPK activity and monocytic differentiation in these cell fractions.

Granulocytes↗

PubMed1989

Hexose monophosphate shunt activity in erythrocytes related to cell age.

Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.

Carbon Radioisotopes↗

PubMed1989

Enolase isoenzymes in human cerebral metastasis.

gamma-Enolase [one of the three possible subunits of the dimeric enzyme enolase (EC 4.2.1.11)] has been reported as a marker for human neurons, neuroendocrine cells and tumors derived from these cells. In recent years, however, its presence has been reported in nonneuronal tumors. For employment in the histopathologic diagnosis of tumors of the nervous system, exact knowledge of the enolase isoenzyme patterns occurring in these tumors is a prerequisite. In human gliomas, the presence of varying quantities of gamma-enolase has been demonstrated. The present study examines the enolase isoenzyme pattern in human cerebral metastases of various primary progeny, using electrophoresis of tumor tissue extracts as well as immunohistochemistry. Additionally, a number of primary tumors of nonneuroepithelial tissue was examined by immunohistochemistry. The presence of gamma-enolase was demonstrated in a significant number of brain metastases. A relation between enolase isoenzyme pattern and survival after operation for brain metastasis could not be found. For histopathologic diagnosis of tumors of the adult human central nervous system, analysis of the enolase isoenzyme pattern is not reliable.

Biomarkers, Tumor↗

PubMed1988

Production of a specific antibody against pyruvate kinase type M2 using a synthetic peptide.

The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2-type holoenzyme of both rat and human origin, and did not cross-react with the M1-type isozyme. This was established by immunoblot analysis, both under dissociating and non-dissociating conditions.

Amino Acid Sequence↗

PubMed1988

Subunit-specific phosphorylation of pyruvate kinase in medullary thyroid carcinomas of the rat.

Pyruvate kinase from anaplastic medullary thyroid carcinomas contains predominantly K-type subunits, whereas pyruvate kinase from differentiated medullary thyroid carcinomas consist of M- and K-type subunits in about equal proportion. In order to analyse the incorporation of phosphate in the respective isozymes after endogenous phosphorylation of cytosolic extracts with [32P]ATP, homotetrameric isozymes as well as heterotetrameric hybrids of differentiated tumors were resolved by affinity chromatography on Blue-Sepharose CL-6B and, if necessary, further purified by immunoprecipitation. SDS-polyacrylamide gel electrophoresis of purified isozymes and subsequent autoradiography showed the incorporation of phosphate in the K4-type isozymes, but not in the other isozymes. The phosphorylation appeared to be cAMP-independent and occurred on a serine residue.

Animals↗

PubMed1988

Discrimination between neuronal and glial cell tumours by pyruvate kinase electrophoresis.

Pyruvate kinase is a tetrameric enzyme, of which different isozymes are known (M, K and L). In normal human brain of adults mainly the M4 type is found with little or none of type K4 or the hybrid K3M. In gliomas of adults as well as in children a shift in the synthesis of isozymes of pyruvate kinase occurs from the M-towards the K-type. In these tumours the hybrid K2M2 is not expressed. In gliomas of children only a spur of K2M2 is found. There exists a peculiar difference in isozyme distribution of pyruvate kinase between gliomas on the one hand and neuro-, retino- and medulloblastomas on the other. In the latter group of tumours often the whole isozyme set is observed and in all cases the hybrids K2M2 and KM3 are found. These findings provide enzymological criteria for the discrimination of neuronal tumours from glial-cell tumours.

Cerebellar Neoplasms↗

PubMed1988

Different pathways for deoxyguanosine toxicity in T-lymphocytes of various developmental stages.

The basis of the selective cellular immunodeficiency which occurs in patients with purine nucleoside phosphorylase (PNP) deficiency still is not completely understood. We studied the mechanism of deoxyguanosine (dGuo) toxicity in proliferating lymphoid T-cells of different maturation stage, i.e. in T-cells of adult peripheral blood and cord blood and in CD3+ and CD3- subfractions of thymocytes. The mitogen-induced proliferation of T-cells from peripheral blood and cord blood and of CD3+ and CD3- subfractions of thymocytes. The mitogen-induced proliferation of T-cells from peripheral blood and cord blood and of CD3+ thymocytes, as well as the spontaneous proliferation of CD3- thymocytes, are inhibited by dGuo. CD3+ and CD3- thymocytes are significantly more sensitive to dGuo than T-cells from peripheral blood or cord blood. Among the thymocyte subfractions CD3- thymocytes appeared to be extremely sensitive. In all cell types studied, inhibition of proliferation is accompanied by intracellular increases in both guanosine triphosphate (GTP) and deoxyguanosine triphosphate (dGTP) concentrations. By use of the PNP inhibitor 8-aminoguanosine, or the metabolites hypoxanthine or deoxycytidine, the metabolism of dGuo could be selectively directed to the formation of GTP or to dGTP. Based on the pattern of rescue from dGuo intoxication under these different metabolic conditions we conclude that in CD3- thymocytes dGuo toxicity is mediated by dGTP. In all other cell types studied GTP mediates dGuo intoxication. Altogether the results show that during the maturation from immature thymocytes to mature peripheral blood T-cells a shift occurs in the pattern of dGuo toxicity since dGuo toxicity in the former is primarily caused via the dCyd kinase pathway, and in the latter mainly the degradation route is involved. Since in PNP deficiency mature T-cells do occur in the peripheral blood, we must conclude that some cells escape the stage of T-cell maturation in the thymus which is extremely sensitive to dGuo. Furthermore, the results imply that as far as T-cell development in the normal thymus is concerned, survival and death of cells might be regulated by local (deoxy) nucleoside availability.

Adult↗

PubMed1988

Alterations in size, shape and osmotic behaviour of red cells after splenectomy: a study of their age dependence.

In whole blood from splenectomized subjects (n = 20), red cells showed a significant increase of mean surface area (MSA), mean cell volume (MCV), MSA/MCV-ratio and osmotic resistance, with the mean cell haemoglobin concentration (MCHC) being decreased. Studies on red cell populations of different cell age revealed that the increase of MSA affects younger and older cells, whereas the increase of MCV can mainly be ascribed to young cells with low density. The increased osmotic resistance is mainly determined by older cells due to a more favourable MSA/MCV-ratio. Shortly after splenectomy (n = 5) the MSA of younger and older cells increased, whereas the increase of MCV affected only young cells with a lowered density; moreover, the MSA/MCV-ratio increased in older cells in particular, resulting in a relatively greater increase of osmotic resistance. An impaired maturation of the reticulocyte may underlie the initial increase of MSA and MCV of young cells, but the present results contradict the current view that delayed maturation explains the changes in morphology and osmotic resistance of asplenic red cells.

Adult↗

PubMed1988

Expression of deoxyadenosine and deoxyguanosine toxicity at different stages of lymphocyte activation.

We have previously shown that deoxyguanosine (dGuo) is toxic to normal T and B lymphocytes, an effect mediated by intracellular accumulation of guanine ribonucleotides. In order to define the cellular processes that are sensitive to guanosine triphosphate (GTP) we have performed studies in which the effects of dGuo on normal T cells are compared with those of deoxyadenosine (dAdo) on adenosine deaminase (ADA)-deficient T cells. Kinetic studies show that dAdo exerts its toxic effects on processes that precede the onset of DNA synthesis, like interleukin 2 receptor expression, whereas dGuo added as late as 24-48 h after initiation of the culture still inhibits mitogen-induced proliferation. It can thus be concluded that dGuo toxicity as mediated through guanine ribonucleotides is manifested relatively late during the process of T-cell activation, whereas dAdo acts early in T-cell activation by a mechanism that cannot be explained by inhibition of ribonucleotide reductase.

Adult↗

PubMed1988

Vacuoles in red cells from splenectomized subjects originate during cell life: association with glycosylated haemoglobin?

Whole blood from splenectomized subjects (n = 8) contained a substantial percentage of vacuolated ('pitted') red cells (34.7 +/- 8.3%), while blood from controls revealed none. The percentage of haemoglobin A1 (HbA1) had increased significantly compared with controls (p less than 0.01). Fractionation on cell density revealed that the number of 'pitted' cells and the HbA1 percentage were associated with increased cell density. Fractionation on cell volume showed that 'pitted' cells are equally distributed in fractions with varying mean cell volume (MCV) and that decreasing MCV was associated with a linear rise of HbA1. It appeared that, shortly after splenectomy (n = 4), 'pits' develop early in cell life and that also older cells, after previous splenic contact, are capable of pit formation. A positive correlation found between the number of 'pitted' cells and the percentage of HbA1 might point to an impaired release of HbA1, manifest in the presence of 'pits'. The increased percentage of HbA1 in whole blood from splenectomized subjects may thus be explained.

Adult↗

PubMed1988

Reticulocyte crisis after splenectomy: evidence for delayed red cell maturation?

In whole blood from splenectomized subjects (n = 8) in a steady state an increased number of reticulocytes was observed (14.0 +/- 7.8% versus 3.6 +/- 2.4% controls: p less than 0.05). Cell fractionation on density showed that reticulocytes, as in normals, are more or less confined to the least dense fraction; cell fractionation on volume revealed that reticulocytes form a heterogeneous cell population with diverging volume, but sharing a low density. Immediately after splenectomy (n = 4), a reticulocytosis is observed, mainly manifest in the top fractions (262.0 +/- 49.0% versus 40.0 +/- 32.4% preoperatively; p less than 0.01). This reticulocytosis is not due to pre- or postoperative complications, but is associated with the splenectomy itself and probably is the consequence of a delayed, but ultimately occurring red cell maturation. The increase of reticulocytes is not accompanied by significant alterations in cell morphology, as far as cell volume is concerned.

Adult↗

PubMed1988

Heterogeneity of glycolytic enzyme activity and isozyme composition of pyruvate kinase in breast cancer.

In 6 patients with breast cancer - of whom specimens of the primary tumor as well as one of its metastases were available for examination - we demonstrated intratumoral and intertumoral heterogeneity in expression of activity of the glycolytic enzymes hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase. Heterogeneity also existed in isozyme composition of pyruvate kinase. The transition of the tumors towards normal surrounding breast tissue showed either a sharp drop in activity, or a gradual decrease in activity, corresponding to pushing margins or infiltrative growth of the tumor as was demonstrated by histologic examination of these specimens. Likewise, the shift towards expression of K isozyme of pyruvate kinase in breast cancer compared to normal breast tissue could be demonstrated.

Adult↗

PubMed1988

Glycolytic enzyme activities in breast cancer metastases.

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Aged↗

PubMed1988

The pyruvate kinase isoenzyme shift in human gliomas: a potential marker in the treatment of gliomas.

In primary human brain tumours a shift occurs in the synthesis of isoenzymes of pyruvate kinase from the M towards the K-type. In astrocytomas, oligodendrogliomas and glioblastomas, which were localised in the cerebral hemispheres of adult patients over 20 years of age, the shift correlated well with histological grading and growth rate as observed in postoperative survival. Gliomas of adults, localised in midline structures, as well as childrens gliomas were characterised too by a strong shift from M towards the K type. However, in these tumours, less correlation with histological grading and growth rate was found. The isoenzyme shift can be rapidly demonstrated with an alanine inhibition test. The application of this assay may have a diagnostic value during operation for gliomas in grading of malignancy in adults as well as demarcation of the resection of gliomas in all age groups. The test can be performed within 10-15 min and can thus fit easily into a surgical procedure. A case report is presented for illustration.

Adolescent↗

PubMed1988

Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase.

The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a "high-ATP syndrome" by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased.

2,3-Diphosphoglycerate↗

PubMed1988

[Value and limitations of the study of gamma-enolase in cerebrospinal fluid as evidence of neuronal degeneration].

Elevation of the gamma-chain of the glycolytic enzyme enolase in the cerebrospinal fluid has previously been reported as a parameter for neuronal damage. The authors evaluated this by measuring gamma-enolase in the CSF of a number of patients with neurological disease known to produce neuronal damage, using a commercial radioimmuno-assay test kit. All measurements were done when gamma-enolase levels had been reported to be maximal during the course of the disease. The results suggest that, as a parameter for neuronal damage, gamma-enolase is not as reliable as reported. An elevation of gamma-enolase in the CSF seems to reflect serious disease; this could be important in differentiating encephalitis caused by Herpes Simplex virus from other viral encephalitides. A normal level of gamma-enolase does not exclude severe neuronal damage.

Adolescent↗

PubMed1988

Production and characterization of monoclonal antibodies against human type K pyruvate kinase.

K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.

Antibodies, Monoclonal↗