Reduction of nitroxide free radical by normal and G6PD deficient red blood cells

The electron spin resonance signal of Tempol decays in the presence of red cells. The decay is due to reduction of oxidant, paramagnetic nitroxide group by the metabolic activity of the red cell. In normal red cells, GSH level was stable and Tempol reduction rate followed a first-order kinetics. In G6PD-deficient red cells, GSH dropped and Tempol reduction rate was slower and followed a second-order kinetics. In normal red cells, diamide reversibly oxidized GSH. First-order kinetics of Tempol reduction rate was attained after a delay time proportional to the diamide concentration and corresponding to the full regeneration of GSH. In diamide-treated G6PD deficient, and in NEM-treated, normal red cells, irreversible disappearance of GSH was followed by irreversible dose-dependent decrease in Tempol reduction rate. A correlation between GSH levels and Tempol reduction rate was observed. A correlation was also established between Tempol reduction rate and stimulation of pentosephosphate shunt activity.     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants KM for glucose-6-phosphate and for NADP+ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP+ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.     

Effect of membrane cholesterol on dimyristoylphosphatidylcholine-induced vesiculation of human red blood cells

During incubation of intact human erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) vesicles, the cells change their discoid morphology to form echinocytes and finally give rise to the release of membrane vesicles. In this process, the red cell membrane accumulates DMPC and loses up to 15% of its cholesterol. On the other hand, replacement of 25% of the endogenous phosphatidylcholine species by DMPC without affecting the cholesterol level of the erythrocytes can be achieved by incubation with DMPC/cholesterol (1:1, mol/mol) sonicated vesicles in the presence of the phosphatidylcholine-specific phospholipid-transfer protein from bovine liver. This replacement also gives rise to an echinocytic cell morphology, but no membrane vesiculation can be observed. However, the vesiculation process can as yet be initiated upon a subsequent decrease of the cholesterol level, by incubation of those modified cells in the presence of sonicated vesicles of pure egg phosphatidylcholine. Incubation of native erythrocytes with pure egg phosphatidylcholine vesicles, on the other hand, results in cholesterol depletion, but does neither induce the formation of echinocytes nor the release of membrane vesicles. Cellular ATP levels are not affected during these incubations. From these results, it can be concluded that a decrease in cholesterol content of the erythrocyte membrane is essential for the DMPC-induced vesiculation of those cells.     

Thrombophilic gene polymorphism studies in G6PD deficient individuals from Saudi population

We performed a study to evaluate the role of three single nucleotide polymorphisms (SNPs), factor V Leiden G1691A (FVL), prothrombin gene mutation G20210A (PRT or FII-G20210A) and methylenotetrahydrofolate reductase variant C677T (MTHFRC677T), as risk factors for G6PD in Saudi populations. Our results did not show any association with the three Thrombophilic genes with FVL gene, no statistical analysis have shown any association with either allele or genotype frequencies OR=0.566, p=.0.667, (95% CI=0.014-22.48) and OR=0.569, p=0.251¸ (95% CI=0.014-22.96).In PRT gene G20210A for G Vs A, p=0.774; OR=0.566 (95%CI; 0.011-29.6); AA+GA Vs GG; p=0.502; OR=0.569 (95%CI=0.010-2969). G and A allele frequencies were similar between cases and controls with no statistical significance. In the MTHFR gene none of the genotypes or allele frequency cannot show any association OR=1.281, p=.0.667, (95% CI=0.414-3.958) and OR=1.1.172, p=0.800¸ (95% CI=0.343-4.008). Similarly, the difference of T allele frequencies between patients and controls was not found any association. In conclusion, our finding indicates that the prevalence of G1691A, G20210A and C677T mutations in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD is not associated with these thrombophilic mutations in Saudi population.     

Interleukin-6-induced tyrosine phosphorylation of phospholipase C-gamma1 in PC12 cells

Phospholipase C (PLC)-gamma1 plays a pivotal role in the signal transduction pathway mediated by growth factors. In this study, we found that neurite outgrowth of pheochromocytoma (PC12) cells was significantly induced by interleukin-6 (IL-6). Stimulation of PC12 cells with IL-6 led to tyrosine phosphorylation of PLC-gamma1 in a dose- and time-dependent manner. IL-6 stimulation also increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Accumulation of total inositol phosphate as well as tyrosine phosphorylation of PLC-gamma1 was inhibited by the pretreatment of protein kinase inhibitors such as genistein and staurosporine. These results suggest that PLC-gamma1 may be involved in the signal transduction pathway of IL-6-induced PC12 cell differentiation.     

Distribution of G6PD phenotypes in red blood cells of Southern African Negroids: Evidence for somatic selection

Summary In a recent population study, we observed a striking deficit of G6PD heterozygotes among Southern African Negroid females. This finding was interpreted tentatively as evidence for a small number of hematopoetic stem cells in man. In a follow-up study we examined peripheral blood and cord blood in 547 mothers and in their newborn offspring. In mothers and sons, the frequencies of the G6PD alleles are apparently quite different. When the allele frequencies determined in sons are used for calculation of the expected phenotype frequencies in mothers and daughters, there is a large deficit of maternal G6PD AB phenotypes, and an equivalent surplus of G6PD homozygotes. However, no relevant heterozygote deficit is observed in newborn daughters. This discrepancy may be explained by the assumption that in peripheral blood of heterozygotes carrying the Gd^A- allele, G6PD-deficient cells progressively become eliminated during development from birth to adulthood. In other words, the large heterozygote deficit observed in adult females may be due to somatic selection rather than to a small pool of hematopoetic cells at the time of X differentiation.H-.H.R. is supported by the Deutsche Forschungsgemeinschaft     

Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol

Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx(m2) ). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx(m2) with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx(m2) -mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxC113S exchange in Trx(m2) . Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes.     

G6PD deficient cells and the bioreduction of disulfides: effects of DHEA, GSH depletion and phenylarsine oxide

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.     

Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: mechanism and implication

More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.     

Glutathione stability and oxidative stress in P. falciparum infection in vitro: Responses of normal and G6PD deficient cells

Red cell oxidative stress in P. falciparum infection in vitro was investigated in relation to the G6PD-Malaria hypothesis. Glutathione stability was enhanced in infected red cells; glucose consumption and pentose pathway activity were not different in normal and G6PD deficient cells, although parasite growth was impaired in G6PD deficiency. Evidence for a response to oxidative stress was not found. Infected red cells have glutamate dehydrogenase activity which was not found in uninfected cells. This enzyme provides a separate pathway for the generation of NADPH independent from the pentose shunt. The data suggest that a significant oxidative stress is not present in falciparum malaria and that another mechanism may be operative in G6PD deficiency.     

G6PD Mediterranean accounts for the high prevalence of G6PD deficiency in Kurdish Jews

The Jews of Kurdistan are a small inbred population with a high incidence of -thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Recently, it was reported that the -thalassaemia in this population shows an unusual mutational diversity; 13 different mutations were identified, of which 4 had not previously been observed in any other population. In contrast, we now report that the G6PD deficiency, which has the highest known incidence in the world, and which affects about 70% of males, is almost entirely attributable to a single widespread mutation, G6PD Mediterranean.     

G6PD deficiency in senile cataracts

Summary The incidence of G6PD deficiency among 338 Thai males with senile cataracts was 5.92% while 446 control Thai males gave an incidence of 6.95%. The figures in females were 16.29% and 14% among 201 senile cataracts females and 200 control females respectively. The age of onset of senile cataracts was not different between the G6PD deficient and G6PD normal groups. The findings indicate that, at least in Thailand, G6PD deficiency in general is not a factor in cataractogenesis.     

ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.     

Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.     

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.