Fulvio Palma

Vanadate affects glucose metabolism of human erythrocytes

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons, and G. Pons (1982) Biochim. Biophys. Acta 705, 238-242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.

Rabbit bone marrow glucose-6-phosphate dehydrogenase during erythroid cell development

Studies were carried out on glucose-6-phosphate dehydrogenase (G6P-DH) during the differentiation of rabbit bone marrow erythroid cells. It was found that G6P-DH, although displaying a 7-fold activity decrease, did not change the relative amounts of its three dimeric forms.Using homogeneous enzyme preparations, we observed that from dividing to non-dividing erythroblasts the following properties remained constant: V max dependence on pH and temperature, Km for G6P dependence on pH, heat stability, 2-deoxy glucose-6-phosphate utilization, molecular weight, while the Km for NADP significantly increased in non-dividing erythroblasts. These results indicate that no shift towards the oxidized form of the enzyme and no substantial modifications of the protein take place during cell differentiation.

Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes.

Niemann-Pick disease type A (NP-A) and type B (NP-B) are lysosomal storage diseases (LSDs) caused by sphingomyelin accumulation in lysosomes relying on reduced or absent acid sphingomyelinase. A considerable body of evidence suggests that lysosomal storage in many LSD impairs autophagy, resulting in the accumulation of poly-ubiquitinated proteins and dysfunctional mitochondria, ultimately leading to cell death. Here we test this hypothesis in a cellular model of Niemann-Pick disease type B, in which autophagy has never been studied. The basal autophagic pathway was first examined in order to evaluate its functionality using several autophagy-modulating substances such as rapamycin and nocodazole. We found that human NP-B B lymphocytes display considerable alteration in their autophagic vacuole accumulation and mitochondrial fragmentation, as well as mitophagy induction (for damaged mitochondria clearance). Furthermore, lipid traceability of intra and extra-cellular environments shows lipid accumulation in NP-B B lymphocytes and also reveals their peculiar trafficking/management, culminating in lipid microparticle extrusion (by lysosomal exocytosis mechanisms) or lipophagy. All of these features point to the presence of a deep autophagy/mitophagy alteration revealing autophagic stress and defective mitochondrial clearance. Hence, rapamycin might be used to regulate autophagy/mitophagy (at least in part) and to contribute to the clearance of lysosomal aberrant lipid storage.

Glucose-6-phosphate dehydrogenase activity and protein turnover in erythroblasts separated by velocity sedimentation at unit gravity and percoll gradient centrifugation

This work was undertaken to improve a separation method for preparation of large amounts of erythroid cells of different age with homogeneous and minimal contamination of myeloid cells. Our method was suitably employed in the study of the decay mechanism of glucose-6-phosphate dehydrogenase (G6PDH) during the erythroid cell maturation.Twenty fractions of erythroid cells at different advancing stages of maturation were prepared by fractionating, at unit gravity, bone marrow cells from anaemic rabbit. The specific activity of the G6PDH was assayed and plotted vs the fraction number and the typical sigmoid curve of the activity decay was drawn. The separated cells were then grouped in three sets of fractions following the three phases of the sigmoid curve and the fractions of each set were combined. From the cytochemical analysis of the three main fractions so obtained, we found a 25–30% myeloid cell contamination in the first fraction, while in the other two fractions the myeloid contamination was 10% or less. For this reason we performed a rapid separation of the first fraction on a discontinuous percoll gradient. By this method, the myeloid cell contamination of the first fraction was levelled down to the other two. The fractions, so obtained, (I, II and III in order of increasing cell maturation) showed a four fold decrease of glucose-6-phosphate dehydrogenase activity expressed both per cell number and on protein base. On the contrary the concentration of the total soluble proteins did not change significantly in the three fractions.The three purified cellular populations were used to provide information on the protein turnover of the erythroid cells during their development. We measured, in intact cells, the rate of synthesis and degradation of total proteins and then, in cell lysates, we determined the rate of degradation of G6PDH, purified from rabbit RBC and radiolabeled by reductive methylation with C14-formaldehyde. The rates of proteolysis obtained with total proteins and methyl-G6PDH clearly indicate that the proteolytic machinery of the erythroblasts reduces its activity during the cell maturation.

Comparative study of the cytoplasmic domain of band 3 from human and rabbit erythrocyte membranes.

The cytoplasmic domain of band 3 (cdb3) is thought to play an important role in human erythrocyte aging. In order to investigate the role of cdb3 during rabbit erythrocyte aging, we compared rabbit cdb3 with the corresponding protein from human erythrocyte membranes. We describe a purification procedure for rabbit cdb3 comparing rabbit and human cdb3 on sodium dodecyl sulphate-polyacrylamide electrophoresis, we found fragments of different molecular weights, implying different chymotryptic cuts in the two species. Anti-human antibodies did not bind to the rabbit cdb3; we also noticed structural differences in the protein between the two species, which may also play a role in the aging processes. Rabbit erythrocyte membranes have a higher capacity of binding hemichromes, due to the higher content of band 3. While in rabbit erythrocyte membranes only one binding site for hemichromes (corresponding to cdb3) was found, we confirmed the existence of two binding sites in human membranes. The second binding site probably corresponds to glycophorin, a protein not present in rabbit membranes.

Glucose-1,6-P2 synthesis, phosphoglucomutase and phosphoribomutase correlate with glucose-1,6-P2 concentration in mammals' red blood cells

Glucose 1,6-biphosphate (G1,6P2) was measured in human, pig, cow, rabbit, rat and sheep red blood cells. Mean values are variable among the species and range from 33 to 122 nmol/ml RBC for pig and rabbit erythrocytes, respectively. The activities of G1,6P2 synthase, phosphoglucomutase (PGM) and phosphoribomutase (PRM) have also been assayed in red cell haemolysates of the same species. The correlations between the biphosphate content and the occurrence of the three enzymatic activities have been studied in order to gain an insight into the regulation of the G1,6P2 turnover in mammalian erythrocytes.

Pharmacological doses of melatonin induce alterations in mitochondrial mass and potential, bcl‐2 levels and K+ currents in UVB‐exposed U937 cells

Apoptosis is observed in ‘actively’ dying cells after the exposure to cell stressors such as ultraviolet light irradiation. Since melatonin has been proposed to act under stressful conditions as cell protection factor, in this study we examined the potential of this molecule when used at pharmacological concentrations to control mitochondrial damage and apoptotic signalling of UVB irradiated U937 human leukaemic cells. Moreover, the effect of melatonin treatment on electrophysiological properties and membrane K+ currents of irradiated U937 cells was investigated as functional aspects relevant to the anti‐apoptotic role of melatonin. The general effect is associated with the restoration of mass, number and membrane potential of mitochondria, with a lower caspase activation and bcl‐2 upregulation. In the presence of the caspase inhibitor ZVAD‐Fmk, melatonin seems to drive UVB stressed cells to follow the mitochondrial intrinsic pathway, interfering just at the mitochondrial level. Moreover, treatment with melatonin, as well as ZVAD‐Fmk, prevented the K+ current reduction observed late following the UVB insult application, by sparing cells from death; this result also indicates that the decrease of K+ leakage currents could represent a functional feature of apoptotic process in UV‐exposed U937 cells.

Acetaldehyde Influences Glucose 1,6-Bisphosphate Level of Human Erythrocytes in vitro and in vivo

In intact erythrocytes from normal adults, acetaldehyde, besides inducing metabolite modifications otherwise observed, markedly decreases the glucose 1,6-bisphosphate (G1,6P2) level. Pyruvate rapidly reverses the acetaldehyde effects. Also in vivo, the acetaldehyde that occurs in the blood stream after heavy alcohol intake produces a significant decrease of the erythrocyte G1,6P2 concentration. These changes support the role of 1,3-bisphosphoglycerate as the first substrate in the G1,6P2 synthesis. The significance of the glucose bisphosphate as glycolytic modulator is also discussed.

Action of Acetaldehyde on Glucose Metabolism of Newborn and Adult Erythrocytes

This paper reports the effect of acetaldehyde on erythrocytes (RBC) of human adults and newborns. Acetaldehyde increases glucose consumption in adult RBC, but has no effect on that of newborn RBC. The compound stimulates the hexose monophosphate shunt and decreases the pyruvate production of the two RBC suspensions. In newborn RBC, acetaldehyde slightly modifies triose-P and fructose-1,6-bisphosphate but has no effect on the ATP/ADP ratio and glucose-1,6-bisphosphate content, which change markedly in adult RBC. Analysis of aldehyde dehydrogenase reveals nearly one half of enzyme activity in newborn RBC. The data indicate that in both adult and newborn RBC, acetaldehyde causes an intracellular reduced state, but the newborn cells take advantage of their greater pyruvate production for complete removal of the exceeding NADH equivalents.

Red cell metabolism affects lactate and pyruvate partition across the plasma membrane

The influence on red cell metabolism of increasing the glucose concentration or the pH of the medium has been examined in order to compare the modification of the lactate/pyruvate ratio inside and outside the cell. In both situations, we evidenced a constancy in the intracellular lactate/pyruvate ratio and an increase in the extracellular one, thus suggesting that the cell efficiently opposes cytoplasmic modifications. In fact an increase of lactate efflux takes place when the intracellular pyruvate decreases. The implications of these changes in the two compartments are discussed on the basis of the available results.