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Biomedical subjects

G E Staal

Publications and source records attributed to G E Staal.

At least 91 records · Page 5Linked to original sources
PubMed1985

Counterflow centrifugation of red cell populations: a cell age related separation technique.

Red cell populations were fractionated on the basis of differences in density by a centrifugation method described by Murphy and on the basis of differences in mean cell volume (MCV) by counterflow centrifugation. By 59Fe-incorporation and determination of the HbA1c content, both methods were studied for their ability to separate red cell populations into fractions of different mean cell age. It can be concluded that separation on the basis of differences in cell volume results in a linear separation according to age whereas separation on the basis of density only results in an accumulation of very young cells in the top fractions. A gradual decrease in cell volume with age, combined with a constant haemoglobin concentration in the cells, indicates release of haemoglobin from the red cells during their lifespan.

Cell Separation↗
PubMed1985

Membrane characteristics and osmotic fragility of red cells, fractionated with anglehead centrifugation and counterflow centrifugation.

Red cell populations were separated on the basis of differences in density using anglehead centrifugation and on the basis of differences in mean cell volume using counterflow centrifugation. In the different fractions, mean surface area was calculated, phospholipid and cholesterol content determined as well as the osmotic behaviour in hypotonic salt solutions. Older red cells appeared to be more resistant to hypotonic salt solutions, due to favourable surface area to volume ratio.

Cell Separation↗
PubMed1985

Age dependent behaviour of red cell glycolytic enzymes in haematological disorders.

The age dependent behaviour of 11, mainly glycolytic, red blood cell enzymes in 26 patients with various haematological disorders has been investigated after separation of red blood cells by discontinuous density gradient centrifugation. The frequency of enzyme deficiencies in the old cells of these patients was significantly increased in comparison with the unseparated cells, 29 and 13 deficiencies, respectively. Particularly hexokinase activity, although normal or even increased in unseparated cells, was found deficient in old cells in seven cases. In addition, an increased number of phosphofructokinase deficiencies was observed in the patients' old cells (eight cases) as compared to the unseparated cells (three cases). However, the red blood cells of the majority of these patients were found to contain increased enzyme activities, irrespective of cell age. Enzyme activities in the youngest cell population did not correlate with the reticulocyte count. Cases of high pyruvate kinase and hexokinase activities were studied for kinetical, electrophoretical and immunological properties of the respective enzymes, but no abnormalities could be demonstrated, indicating an increased synthesis of these enzymes.

Centrifugation, Density Gradient↗
PubMed1985

Isozymic composition and regulatory properties of phosphofructokinase from well-differentiated and anaplastic medullary thyroid carcinomas of the rat.

Acceleration of glycolysis is, in general, a characteristic of neoplasia. Previous studies have shown that this increase in glycolysis is achieved by quantitative increases in the activities of the key regulatory enzymes, hexokinase, phosphofructokinase (PFK) and/or pyruvate kinase, which are often accompanied by isozymic alterations that facilitate glycolysis. In this study, we investigated the alterations in the activity, isozymic profile, and kinetic-regulatory properties of PFK from the medullary thyroid carcinomas of the rat, which represent a model for the neuroectodermally derived tumors in humans. Contrary to the expected, we found that undifferentiated tumors showed a decrease in the enzyme activity as compared to the highly differentiated tumors. This decrease in PFK activity was accompanied by an increase in the expression of the liver-type isozyme of PFK. The enzymes from the 2 tumor types showed no significant differences in their affinity and cooperativity toward the substrates, fructose 6-phosphate and adenosine triphosphate (ATP). However, the tumor PFKs showed major differences with respect to their behavior toward the allosteric regulators of the enzymes, ATP, citrate, and fructose 2,6-diphosphate; the latter is a recently discovered activator of the enzyme. The enzyme from the undifferentiated tumor was less sensitive to citrate inhibition, which was more readily reversed by cyclic adenosine 3':5'-monophosphate. In addition, it was less sensitive to ATP inhibition at low fructose 6-phosphate concentrations. More importantly, the enzyme from the undifferentiated tumors was more sensitive to the activation by fructose 2,6-diphosphate especially when inhibited by citrate and ATP. The altered regulatory properties of the enzyme from the undifferentiated tumors most probably reflect its altered isozymic composition, i.e., increase in the liver-type isozyme. The preferential expression of the liver-type isozyme by undifferentiated and rapidly replicating cancer cells may be explained in terms of the unique regulatory properties of this isozyme. Although the concentrations of fructose 2,6-diphosphate were comparable in these 2 tumor types, the higher sensitivity of the liver-type PFK to activation by this compound may permit accelerated glycolytic flux observed in undifferentiated tumors, despite a decrease in total PFK activity.

Animals↗
PubMed1985

Pyruvate kinase isozymes and dedifferentiation of (neuro-)ectodermal tumors.

This paper describes the isozyme composition and regulatory properties of pyruvate kinase (PK) from well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. These tumors were chosen as an animal model for human (neuro-)entodermal neoplasms differing in their degree of differentiation. The results were compared with human medullary thyroid carcinomas (MTC) and phaeochromocytomas. AMTC were characterized by increased PK activity, a higher apparent S0.5 for phosphoenolpyruvate, enhanced inhibition by alanine, presence of predominantly M2-type isozyme and loss of M1-type-containing hybrids as compared to DMTC. The alterations in PK isozyme expression and hence kinetic behaviour could not be demonstrated in human MTC or phaeochromocytomas due to the apparently well-differentiated nature of these tumors and the presence of M2-type isozymes. These results are discussed with reference to the nature and significance of PK isozyme shifts found in other tumors. It is suggested that the determination of PK isozyme composition might prove useful in the diagnosis of nueroectodermal neoplasms originating from tissues not primarily expressing M2-type isozyme(s).

Animals↗
PubMed1984

Isozyme pattern of enolase of childhood tumors.

Retinoblastoma, neuroblastoma, and medulloblastoma have many common features, clinical as well as histologic; a common embryonic origin has been suggested. The authors studied the electrophoretic pattern of enolase (EC 4.2.1.11) in these tumors. All tumors were characterized by the presence of three types of enolase, designated as alpha alpha, alpha gamma and gamma gamma. The latter is supposed to be the neuron-specific enolase. Normal adult brain and adult retina show the same set of isozymes (alpha alpha, alpha gamma and gamma gamma). In contrast, gliomas of childhood, tumors originating from the supportive tissue of the central nervous system, are characterized mainly by the presence of the alpha alpha dimer and a small amount of the alpha gamma hybrid. The results of this report support the hypothesis of a common embryonic origin of retinoblastoma, neuroblastoma, and medulloblastoma.

Adult↗
PubMed1984

Hexokinase, phosphofructokinase and pyruvate kinase isoenzymes in lymphocyte subpopulations.

In order to study the three regulator enzymes of glycolysis, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), in relation to lymphocyte maturation, lymphocytes of different origin were investigated. Lymphocytes from bone marrow, thymus, cord blood, adult peripheral blood and mitogen-stimulated lymphocytes were investigated. The enzyme activities were determined and the isozyme patterns were studied by means of electrophoresis, kinetic measurements and immunoprecipitation. The young lymphocytes from bone marrow and the mitogen-stimulated lymphocytes could be distinguished from the other lymphocytes by a higher residual HK activity in the presence of the inhibitor glucose-1,6-diphosphate. Peripheral blood T lymphocytes differed from non-T lymphocytes in the PK isozymes distribution. All the cells contained PK type K4 and the hybrid K3M. In T cells a smaller amount of the K isozyme was seen than in non-T cells. The PK residual activity in the presence of alanine was significantly higher in peripheral blood T cells than in non-T cells. Thymocytes are characterised by a larger amount of PFK M-subunits than peripheral blood T and non-T lymphocytes. The stimulation of PFK by the positive effector glucose-1,6-diphosphate was higher in thymocytes than in the peripheral blood lymphocytes.

Bone Marrow↗
PubMed1984

Hexokinase isoenzymes from anaplastic and differentiated medullary thyroid carcinoma in the rat.

The activity, isoenzyme distribution and compartmentation of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were compared in slowly growing, well-differentiated medullary thyroid carcinoma (DMTC) and rapidly proliferating anaplastic thyroid carcinoma (AMTC) in the rat. Individual isoenzymes from either soluble or particulate fractions after solubilization were obtained by fast protein liquid chromatography and were kinetically analyzed either in soluble form or after (re)binding to rat liver mitochondria. These studies were undertaken to test the hypothesis that the growth rate of tumors is correlated with the activity of mitochondrial-bound hexokinase in our tumor system. In contradiction to this hypothesis, we found no difference in either enzyme activity or compartmentation of both kinds of tumors. The major part of enzyme activity was soluble (73 and 78% in DMTC and AMTC respectively). In addition, no major differences were observed in the kinetic properties of the individual isoenzymes of both tumors. Only soluble type II hexokinase from AMTC had a slightly decreased apparent Km for glucose. There appeared to be some differences in isoenzyme composition: both tumors contained type I and type II hexokinase in the soluble as well as in the particulate fractions. However, the proportion was shifted in favor of type II hexokinase in the soluble fraction of AMTC. Additional findings of this study were the following: the affinity of type II hexokinase to both substrates glucose and MgATP2- was significantly less compared to type I hexokinase. However, the inhibition constant for glucose-1,6-diphosphate of both isoenzymes was exactly the same. The bound form of both isoenzymes had the same substrate affinities as the soluble form but was considerably less inhibited by glucose-1,6-diphosphate. In the latter respect, type I and type II hexokinase behaved in the same way.

Adenosine Triphosphate↗
PubMed1984

Glycolytic enzymes from human neuroectodermal tumors of childhood.

In this study pyruvate kinase, hexokinase and aldolase are investigated in two types of embryonal tumors, neuroblastomas and medulloblastomas; the results are compared with similar studies in gliomas. The activities of hexokinase and pyruvate kinase are significantly decreased in neuroblastomas. In neuroblastoma and medulloblastoma all five forms of pyruvate kinase (K4, K3M, K2M2, KM3 and M4) are present. In contrast, the gliomas investigated are characterized by the presence of mainly K4 and a little K3M. In neuroblastomas, medulloblastomas and gliomas, hexokinase type I is present; in addition, hexokinase type II is present in two medulloblastomas. Aldolase A is the predominant isozyme in all tumors investigated; this is in contrast with normal nervous tissue. It can be concluded that the isozyme characteristics especially of pyruvate kinase from neuroblastomas and medulloblastomas are comparable with similar findings in retinoblastoma; these findings support the hypothesis that these three tumors have a common embryonic origin.

Child↗
PubMed1984

Pyruvate kinase and the "high ATP syndrome".

The erythrocytes of a patient with the so-called "high ATP syndrome" were characterized by a high ATP content and low 2,3-diphosphoglycerate level. The pyruvate kinase activity was specifically increased (about twice the normal level). After separation of the erythrocytes according to age by discontinuous Percoll density centrifugation, the pyruvate kinase activity was found to be increased in all Percoll fractions. Pyruvate kinase of the patient's cells was characterized by a decreased K0.5 for the substrate phosphoenolpyruvate and no inhibition by ATP. The Michaelis constant (Km) value for ADP, the nucleotide specificity, the thermostability, pH optimum, and immunological specific activity were normal. It is concluded that the high pyruvate kinase activity is due to a shift in the R(elaxed) in equilibrium T(ight) equilibrium to the R(elaxed) form.

2,3-Diphosphoglycerate↗
PubMed1984

The effect of deoxyguanosine on human lymphocyte function. I. Analysis of the interference with lymphocyte proliferation in vitro.

The effect of deoxyguanosine on mitogen- and antigen-induced proliferation of peripheral blood lymphocytes from healthy donors was studied. Deoxyguanosine was found to inhibit the proliferative response to mitogens and antigens. Concentrations of deoxyguanosine causing 50% inhibition of the proliferation proved to be dependent on the activity of catabolic enzymes, such as purine nucleoside phosphorylase (PNP), in sera used in the culture media. The inhibitory effect of deoxyguanosine on phytohemagglutinin (PHA)-induced cell proliferation was prevented by deoxycytidine as well as by hypoxanthine. These findings were analyzed further by determination of intracellular (deoxy)-nucleotide levels. Stimulation of lymphocytes by PHA in the presence of deoxyguanosine leads to intracellular accumulation of dGTP. The presence of hypoxanthine in addition to deoxyguanosine abolished the inhibitory effect but did not prevent dGTP accumulation. On the other hand, the addition of deoxycytidine in combination with deoxyguanosine did not lead to intracellular accumulation of detectable amounts of dGTP, but only gave partial protection against the toxic effect. Furthermore, guanosine inhibited mitogen-induced cell proliferation to the same extent as did deoxyguanosine provided that the culture media were supplemented with pretreated fetal calf serum. Peripheral blood lymphocytes of a PNP-deficient or a HGPRT-deficient patient in cultures stimulated with PHA or pokeweed mitogen were resistant to the inhibitory effects of guanosine and were less sensitive to deoxyguanosine than cells of normal donors. The present results clearly show the involvement of two pathways contributing to deoxyguanosine-mediated inhibition of the proliferation of normal lymphocytes, i.e., on the one hand degradation of deoxyguanosine by PNP, salvage of guanine by HGPRT, and (possibly) phosphorylation of GMP eventually leading to GTP, and on the other hand formation of dGTP by direct phosphorylation of deoxyguanosine by deoxycytidine kinase.

Deoxycytidine↗
PubMed1984

The effect of deoxyguanosine on human lymphocyte function. II. Analysis of the interference with B lymphocyte differentiation in vitro.

The differentiation of normal human peripheral blood B lymphocytes into plasma cells in vitro, studied in mononuclear cells stimulated with PWM or in purified B cells stimulated with a T cell-replacing factor (TRF), can be inhibited by both deoxyguanosine (dGuo) and guanosine. The mechanism underlying this effect, which differs from the in vivo findings in PNP deficiency, was analyzed. dGuo toxicity can be antagonized by hypoxanthine but not by deoxycytidine. PNP-deficient and HGPRT-deficient B lymphocytes are not sensitive to the intoxicating properties of (deoxy)guanosine. Inhibition of PNP activity in normal B lymphocytes by 8-aminoguanosine decreases the sensitivity for dGuo intoxication. Incubation of purified B cells (stimulated with TRF) with dGuo leads to increased intracellular levels of guanosine di- and triphosphate (GDP and GTP), whereas deoxyguanosine triphosphate (dGTP) levels remain low. These observations lead to the conclusion that inhibition of B lymphocyte differentiation by dGuo is brought about by one of the end products of the pathway starting with degradation of dGuo by PNP, followed by guanine salvage by HGPRT, and possibly further phosphorylation of GMP into GDP and GTP. According to this mechanism, B lymphocyte differentiation in PNP deficiency is not sensitive to (deoxy)guanosine; because of the absence of PNP activity, these cells cannot accumulate GMP, GDP, and GTP, and therefore escape dGuo intoxication.

B-Lymphocytes↗
PubMed1984

Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro.

Late committed progenitor cells of erythropoiesis, CFU-E (colony-forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6-phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU-E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU-E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

Animals↗
PubMed1984

Glucose-6-phosphate isomerase deficiency-Nahariya: extreme in vitro and in vivo lability of the mutant enzyme.

A glucose-6-phosphate isomerase deficiency is described in an Arab boy suffering from chronic hemolytic anemia. The patient was probably true homozygous for the defect. The residual enzyme activity in his red blood cells (RBC) was approximately 30% of normal. The most striking enzyme abnormality observed was an extreme heat lability: upon incubation at 45 C, greater than 90% of activity was lost within 15 min. Furthermore, an increased affinity for the substrate glucose-6-phosphate was shown. The lability of the enzyme was also shown to exist in vivo by separating the patient's RBC into four fractions of different cell age by centrifugation on a discontinuous density gradient. This in vivo lability of the enzyme is believed to be the main cause of the hemolytic diathesis. Remarkably, the residual activity of the enzyme in the RBC of obligate heterozygotes was comparable to that in the patient. However, their enzyme activity was only slightly more labile than that in normal RBC and consequently no signs of hemolysis were noticed.

Anemia, Hemolytic↗