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M Magnani

Publications and source records attributed to M Magnani.

At least 181 records · Page 10Linked to original sources
PubMed1989

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes.

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.

2,3-Diphosphoglycerate↗
PubMed1988

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Adenine Nucleotides↗
PubMed1988

Hexokinase type I multiplicity in human erythrocytes.

Hexokinase I in human erythrocytes exists in multiple molecular forms that differ in isoelectric points. By means of Western blotting and immunodetection of total glucose-phosphorylating activity by using an antibody raised in rabbit against homogeneous human placenta hexokinase I, a single protein band was detected. Identical results were also obtained by immunoaffinity chromatography of the partially purified enzyme. Separation of the three major hexokinase I subtypes (Ia, Ib and Ic) by h.p.l.c. ion-exchange chromatography and immunodetection following electrophoretic blotting confirmed that each hexokinase subtype showed the same apparent Mr of 112,000, which is the value obtained for the high-Mr hexokinase I from human placenta. Purification of erythrocyte hexokinase by a combination of several procedures including dye-ligand and affinity chromatography that were previously successfully applied to the purification of other mammalian hexokinases type I produced a 35,000-fold-purified enzyme that showed several contaminants after SDS/polyacrylamide-gel electrophoresis. Only one of these peptides was found to be recognized by anti-(hexokinase I) IgG, suggesting that proteolytic degradation does not occur and that hexokinases Ia, Ib and Ic have the same apparent Mr.

Chromatography, High Pressure Liquid↗
PubMed1988

Role of hexokinase in the regulation of erythrocyte hexose monophosphate pathway under oxidative stress.

Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.

Erythrocytes↗
PubMed1988

The interaction of phosphorylated sugars with human hexokinase I.

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.

Adenosine Triphosphate↗
PubMed1988

Effect of phenylhydrazine on red blood cell metabolism.

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.

Adenine Nucleotides↗
PubMed1988

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation.

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase Ia can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during 'in vivo' reticulocytes maturation. This new hexokinase microheterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.

Animals↗
PubMed1988

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I.

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.

Centrifugation, Density Gradient↗
PubMed1988

Effect of age on some properties of mice erythrocytes.

The hematological parameters of young (2-month-old) and old (2-year-old) mice were compared. No differences could be detected with the exception of an increased percentage of reticulocytes in the old animals suggesting that anemia in senescent mice does not occur. Red blood cell mean half-life in old mice was 8 +/- 0.8 days compared to 12 +/- 1 days in young mice. This reduced survival of red blood cell is not due to a different rate of cell phagocytosis in the reticulohistiocytic system of young and old animals since erythrocytes from young mice have the same mean half-life when injected both in young and old animals and vice versa. Thus, the old mice have a reduced red cell life-span but the same hematocrit of the young, suggesting that old animals possess a chronologically younger population of erythrocytes than do young animals. This has been confirmed by measuring the specific activities of some red blood cell age-dependent enzymes (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase) that were found to be higher in the older animals, and by the separation of erythrocytes into different density (age) groups by Percoll/albumin density gradient centrifugation. However, the erythrocytes osmotic fragility, and the cellular contents of adenine and pyridine nucleotides, as well as the content of 2,3-diphosphoglycerate and reduced glutathione, show that circulating erythrocytes in old animals constitute an heterogeneous cell population whose properties cannot be explained on the basis of a chronologically younger erythrocyte population. Furthermore, evaluation of cell components in hemopoietic tissues have shown an increased porportion of erythroid precursor cells in old animals confirming that old mice compensate for reduced red cell survival with an increased erythropoiesis.

Aging↗
PubMed1988

Simultaneous preparation from human placenta of several enzymes of glucose metabolism.

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.

Chromatography, Affinity↗
PubMed1988

Muscarinic modulation of purine release from electrically stimulated rat cortical slices.

The release of 3H-labeled purines at rest and during electrical stimulation was investigated in slices of rat cortex prelabeled with [3H]adenine and perfused with Krebs solution. A linear relationship was found between radioactivity efflux and stimulation frequency from 2.5 to 20 Hz. At frequencies of less than 2.5 Hz, no increase in radioactivity efflux was detected. The amount of tritium released per pulse increased with stimulation frequency up to 10 Hz and declined at 20 Hz. The tritium efflux from the slices at rest and at a stimulation frequency of 10 Hz, analyzed by HPLC with ultraviolet absorbance detection at 254 nm, consisted mostly of adenosine, inosine, and hypoxanthine. The 3H-labeled purine release evoked by 10-Hz stimulation increased with current intensity from 15 to 100 mA/cm2. At 20 mA/cm2, addition of 0.5 microM tetrodotoxin to the superfusing Krebs solution brought about a 98% decrease of 3H-labeled purine release. At higher current strength, the percentage of tetrodotoxin-sensitive-evoked tritium efflux was smaller. At 30 mA/cm2, 86% of the evoked release was tetrodotoxin sensitive. Under these stimulation conditions, tritium efflux showed a 69% decrease when the slices were superfused with calcium-free Krebs solution containing 0.5 mM EGTA. The muscarinic agonist oxotremorine (30 microM) significantly enhanced the 10-Hz-stimulated 3H-labeled purine release. The effect of oxotremorine was partially prevented by tetrodotoxin, was antagonized by atropine (1.5 microM), and was mimicked by addition of physostigmine (3.8 microM) to the superfusion fluid. Atropine alone did not affect the evoked release, and none of the drugs modified the basal tritium efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1988

Membrane-bound immunoglobulins increase during red blood cell aging.

A flow cytofluorimetric method was used to detect the immunoglobulin molecules present on human red blood cells. Normal human erythrocytes were separated into seven fractions of increasing mean age by density centrifugation on discontinuous gradients. Some biochemical and morphological properties of these cells were determined as well as their IgG content. The results obtained suggest that IgG binding during red cell aging is a cumulative process.

Erythrocyte Aging↗
PubMed1987

An analysis of cholecystokinin-induced increase in acetylcholine output from cerebral cortex of the rat.

The effect of colecystokinin (CCK-8) on the release of ACh from the cerebral cortex was studied in urethane-anaesthetized rats with the cortical cup technique. The increase in output of ACh brought about by the administration of CCK-8 1.5 micrograms/kg (i.p.) was prevented by pretreatment with haloperidol (1 mg/kg i.p.) and by lesions of the nucleus basalis magnocellularis and substantia nigra but it was reduced only slightly by bilateral vagotomy. Conversely, none of the treatments abolished the decrease in output of ACh brought about by CCK-8 at a dose of 10 micrograms/kg (i.p.). Local injection of CCK-8 into the nucleus basalis had no effect. Therefore, CCK-8 appears to increase cortical cholinergic activity by indirectly stimulating the cholinergic neurones of the nucleus basalis through dopaminergic neurones.

Acetylcholine↗
PubMed1987

Effect of pyroglutamic acid stereoisomers on ECS and scopolamine-induced memory disruption and brain acetylcholine levels in the rat.

The acquisition of a passive avoidance conditioned response was disrupted in the rat by electroconvulsive shock (ECS) and scopolamine administration. D,L-pyroglutamic acid (D,L-PCA) 500 and 1000 mg/kg, administered as arginine salt 120 min before the retest, prevented both the ECS and scopolamine-induced amnesia. Arginine alone was ineffective. Scopolamine brought about a 52 and 39% decrease, respectively, in cortical and hippocampal acetylcholine (ACh) levels, measured by means of a gas-chromatographic method. D,L-PCA 500 and 1000 mg/kg also prevented the decrease in brain ACh level. When the two isomers were studied separately, D-PCA was more effective than L-PCA and antagonized scopolamine-induced amnesia at the doses of 250 and 500 mg/kg. In conclusion, D,L-PCA is active on cortical and hippocampal cholinergic mechanisms and, like other 2-oxopyrrolidone derivatives, shows cognition-enhancing properties.

Acetylcholine↗
PubMed1987

Adenine and pyridine nucleotides during rabbit reticulocyte maturation and cell aging.

In this paper we have used a new method which allows the simultaneous extraction and HPLC determination of ATP, ADP, AMP, NADP, NADPH, NAD and NADH to evaluate the changes in concentration of these compounds during maturation of rabbit reticulocytes and cell aging. The results show a significant increase of ATP concentration, higher ATP/ADP, ATP/AMP ratios and lower NADP+/NADPH, NAD+/NADH ratios in rabbit reticulocytes when compared to mature cells. Similar results were also obtained when the whole red blood cell population was separated into fractions of increasing mean age on discontinuous gradients of Percoll-BSA. These metabolic modifications are discussed in relation to the age-dependent metabolic decline of the erythrocyte.

Adenine Nucleotides↗
PubMed1987

Immunohistochemical determination of growth fractions in human permanent cell lines and lymphoid tumours: a critical comparison of the monoclonal antibodies OKT9 and Ki-67.

OKT9 and Ki-67 monoclonal antibodies have recently been proposed as useful tools for evaluating the growth fraction in malignant tumours, with special reference to non-Hodgkin's lymphomas. In particular, while the former is commonly thought to detect a transferrin receptor present on the cytoplasmic membrane of proliferating cells, the latter recognizes a nuclear antigen, which is expressed in G1, S, G2 and M phase of continuously cycling elements. To further verify their reliability, OKT9 and Ki-67 were applied to seven permanent cell lines (four myeloid and three B-lymphoblastoid) and 100 lymphoid tumours (70 non-Hodgkin's and 30 Hodgkin's lymphomas) phenotypically characterized on frozen sections. The results obtained showed that OKT9 and Ki-67 cannot be employed as equivalent means in assessing the growth fraction. In fact, OKT9 is directed to a transferrin receptor which is not only expressed by proliferating cells, but also by some resting elements. On the other hand, Ki-67 provides a nuclear, easily detectable positivity which is restricted to proliferating cells only. Therefore, it seems to represent the only monoclonal which can confidently be employed in the assessment of the growth fraction. Furthermore, the present study underlines that the immunocytochemical analysis of the proliferation rate in tumours gives similar information to the radionucleide uptake assay, while it represents a more sensitive method than the cytofluorimetric evaluation of the DNA content.

Antibodies, Monoclonal↗
PubMed1987

Red blood cell glucose metabolism in Down's syndrome.

The specific activity of red blood cell glycolytic enzymes was determined in 20 Down's syndrome patients and compared with 20 normal controls. According to previous evidence, a 50% increase of phosphofructokinase and a 30% increase of glucose-6-phosphate dehydrogenase and glutathione peroxidase activity was found. Metabolic studies of the patients' erythrocytes revealed a decrease in fructose-6-phosphate and 2, 3-diphosphoglycerate concentrations, while fructose-1, 6-diphosphate and ADP both increased. Glucose utilization by intact erythrocytes from Down's syndrome patients did not differ from that of normal controls. However, addition of methylene blue or inorganic phosphate produced a higher stimulation of erythrocyte glycolysis in patients with Down's syndrome compared to controls. These metabolic abnormalities could be, at least in part, ascribed to the increased phosphofructokinase activity which is due to a gene-dosage effect.

Blood Glucose↗