Giuseppe Martini

Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene- deleted mouse embryonic stem cells subjected to oxidative stress

Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells ( G6pd delta), obtained by transient Cre recombinase expression in a G6pd -loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pd delta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pd delta cells show a high ratio of NADPH to NADP(+) and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pd delta but not for wild-type ES cells, NADPH and GSH in G6pd delta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP(+)] ratio, does not occur in G6pd delta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.

Molecular anatomy of the human glucose 6‐phosphate dehydrogenase core promoter

The gene encoding glucose 6‐phosphate dehydrogenase (G6PD), which plays a pivotal role in cell defense against oxidative stress, is ubiquitously expressed at widely different levels in various tissues; moreover, G6PD expression is regulated by a number of stimuli. In this study we have analyzed the molecular anatomy of the G6PD core promoter. Our results indicate that the G6PD promoter is more complex than previously assumed; G6PD expression is under the control of several elements that are all required for correct promoter functioning and, furthermore, a still unidentified mammalian specific factor is needed.

2-deoxy-d-ribose induces apoptosis by inhibiting the synthesis and increasing the efflux of glutathione.

Oxidative stress is caused by imbalance between the production of reactive oxygen species (ROS) and biological system ability to readily detoxify the reactive intermediates or repair the resulting damage. 2-deoxy-D-ribose (dRib) is known to induce apoptosis by provoking an oxidative stress by depleting glutathione (GSH). In this paper, we elucidate the mechanisms underlying GSH depletion in response to dRib treatment. We demonstrated that the observed GSH depletion is not only due to inhibition of synthesis, by inhibiting gamma-glutamyl-cysteine synthetase, but also due to its increased efflux, by the activity of multidrug resistance associated proteins transporters. We conclude that dRib interferes with GSH homeostasis and that likely cellular oxidative stress is a consequence of GSH depletion. Various GSH fates, such as direct oxidation, lack of synthesis or of storage, characterize different kinds of oxidative stress. In the light of our observations we conclude that dRib does not induce GSH oxidation but interferes with GSH synthesis and storage. Lack of GSH allows accumulation of ROS and cells, disarmed against oxidative insults, undergo apoptosis.

Molecular basis of chronic non-spherocytic haemolytic anaemia: a new G6PD variant (393 Arg----His) with abnormal KmG6P and marked in vivo instability.

Summary. More than 80 genetic variants of glucose‐6‐phosphate dehydrogenase (G6PD) are associated with chronic non‐spherocytic haemolytic anaemia (CNSHA). In order to help clarify the molecular basis of this association, we have carried out a detailed biochemical and genetic characterization of two G6PD deficient brothers affected by CNSHA. The G6PD from the two patients has altered electrophoretic mobility, abnormally elevated Michaelis constant (Km) for G6P, and extreme instability in vivo and in vitro. By comparison with published information we found that this is a new G6PD variant which we have designated G6PD Portici. The entire coding region of the gene has been sequenced, and a single point mutation, a G→A transition, was found at position 1178 in exon X, causing a substitution of histidine for arginine at residue 393 in the polypeptide chain. By polymerase chain reaction (PCR) amplification followed by diagnostic restriction enzyme analysis and allele‐specific oligonucleotide hybridization we have demonstrated the inheritance of this mutation in the patients family. Our results support the notion of a causative link between this mutation in the G6PD gene and CNSHA. Our data, in combination with previous data in the literature, suggest that the three‐dimensional structure of G6PD is such as to cause interaction in the binding of its two substrates, G6P and NADP.

High levels of transcription driven by a 400 bp segment of the human G6PD promoter

A 2850-base-pair-long DNA segment containing the transcriptional start site of the human X-linked gene coding for the housekeeping enzyme glucose-6-phosphate dehydrogenase has been fused to the reporter chloramphenicol acetyl transferase gene. This fusion was introduced into three different mammalian cell lines (HepG2, CVI and HeLa). Strong expression was detected and transcription initiated at the natural start site. By deletion analysis, we determined that a 436 base pair region of this promoter is sufficient for full expression of the chimaeric construct and therefore the remainder of the CpC island surrounding the transcriptional start site and differentially methylated in the active and inactive X chromosome is not necessary for transcriptional activity in this assay. Further sequence deletions from -436 to -116 base pairs decrease the promoter-directed chloramphenicol acetyl transferase activity. Site-directed mutagenesis of a TATA-like sequence present in the G6PD promoter shows that this site is required for a correct start of transcription but not for determining the level of gene expression.

Molecular characterization of G6PD deficiency in Southern Italy : Heterogeneity, correlation genotype-phenotype and description of a new variant (G6PD Neapolis)

We report on the molecular basis of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty‐one unrelated G6PD‐ deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A−, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A− and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467Pro→Arg substitution reponsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.

High-level regulated expression of the human G6PD gene in transgenic mice

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.

Discussion on Pharmacogenetic Interaction in G6PD Deficiency and Methods to Identify Potential Hemolytic Drugs

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common form of red blood cell enzymopathy. The disorder has reached polymorphic frequencies in different parts of the world due to the relative protection conferred against malaria. G6PD is a housekeeping X-linked gene encoding the first enzyme of the pentose phosphate pathway, an NADPH-producing dehydrogenase. Because erythrocytes do not generate NADPH in any other way than pentose phosphate pathway, they are more susceptible than any other cells to oxidative damages. G6PD deficiency is a prime example of a hemolytic anemia due to an interaction between an intracorpuscular cause and an extracorpuscular cause, because in the majority of cases an exogenous agent triggers hemolysis. Hemolysis, in fact, can be caused by exposure to oxidant agents. Although studies performed on epidemiology, genetics and molecular biology have broaden the information on G6pd deficiency, there are still no reliable and validated methods to test drug hemolytic potential in G6PD deficient patients. The review gives an overview of current knowledge on G6pd deficiency and on the methods that have been developed so far in order to identify drugs causing acute hemolytic anemia in G6pd deficiency. Moreover, we discuss the new potential preclinical strategies to assess, in vitro and in vivo, drug hemolytic risks.

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli.

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.

Fungal extrachromosomal DNA and its maintenance and expression in E. coli K-12

A phytopathogenic fungus, Fusarium oxysporum , has been found to contain a plasmid-like DNA molecule which can be introduced by transformation into E. coli K-12 strains, where it can be maintained as a bacteria plasmid. An E. coli K-12 strain bearing this fungal DNA is able to express a polysaccharide hydrolizing activity which is known to be involved in the fungal pathogenic processes.