Genome-wide Analysis of Glucose-6-phosphate Dehydrogenase(G6PDH) and Its Evolution in Eucalyptus grandsis

[Objective] The aim of this study was to perform genome-wide analysis of glucose-6-phosphate dehydrogenase（G6PDH） and reveal its evolution in Eucalyptus grandsis.[Method] The gene character,protein sequence and phylogenetic tree of G6PDH gene were analyzed by BLAST and other bioinformatics software within Eucalyptus grandsis whole genome database.[Result] Six G6PDH genes,including one cytomic type and five plastids,were detected in the E.grandsis genome.All the G6PDHs have conserved motifs of motif 1,motif 2,motif 3,motif 7,motif 9 and motif 11.Furthermore,promoter sequences of all E.grandsis G6PDH contain TATA box,enhancer,light-responsive,hormone-responsive and stress-responsive regulatory elements.[Conclusion] This study provided reference for the further revealing molecular function of E.grandsis G6PDH gene family

High Level Expression of Glucose-6-phosphate Dehydrogenase Gene PsG6PDH from Populus suaveolens in E. coli

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G （pET30a-G6PDH）, which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol·L^-1 IPTG treatment for 4h and that pET-G product was predominately soluble and not extra-cellular secreting.

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.

Involvement of the circular RNA/microRNA/glucose-6-phosphate dehydrogenase axis in the pathological mechanism of hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is the third most common cause of cancer-related death worldwide with high mortality.The incidence of HCC is increasing in China.Abnormal activation of glucose-6-phosphate dehydrogenase(G6 PD)exists in all malignant tumors,including HCC,and is closely related to the development of HCC.In addition,the differential expression of non-coding RNAs is closely related to the development of HCC.This systematic review focuses on the relationship between G6 PD,HCC,and noncoding RNA,which form the basis for the circ RNA/mi RNA/G6 PD axis in HCC.The circular RNA(circ RNA)/micro RNA(mi RNA)/G6 PD axis is involved in development of HCC.We proposed that non-coding RNA molecules of the circ RNA/mi RNA/G6 PD axis may be novel biomarkers for the pathological diagnosis,prognosis,and targeted therapy of HCC.

Is glucose-6-phosphate dehydrogenase deficiency more prevalent in Carrion's disease endemic areas in Latin America?

Glucose-6-phosphate dehydrogenase(G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention.Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria,giving G6 PD a major role in its stability.G6 PD deficiency(G6PDd) is the most common enzyme deficiency in humans:it affects approximately 400 million individuals worldwide.The overall G6 PDd allele frequency across malaria endemic countries is estimated to be 8%.corresponding to approximately 220 million males and 133 million females.However,there are no reports on the prevalence of G6 PDd in Andean communities where bartonellosis is prevalent.

Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in India: A Systematic Review

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common enzyme deficiency of human erythrocyte affecting more than 400 million people worldwide. In India, G6PD deficiency was first reported in 1963 and since then various investigations have been conducted across country. The objective of this work was to study the prevalence of G6PD deficiency in different ethnic, caste and linguistic groups of Indian population. A systematic search of published literature was undertaken and the wide variability of G6PD deficiency has been observed ranging from 0% - 30.7% among the different caste, ethnic, and linguistic groups of India. It was observed that the incidence of G6PD deficiency was found to be considerably higher among the tribes (9.86%) as compared to other ethnic groups (7.34%) and significantly higher in males as compared to females.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

Glucose-6-phosphate dehydrogenase(G6PD) deficiency is associated with asymptomatic malaria in a rural community in Burkina Faso

Objective:To investigate 4 combinations of mutations responsible for glucose-6—phosphate dehydrogenase(G6PD) deficiency in a rural community of Burkina Faso,a malaria endemic country.Methods:Two hundred individuals in a rural community were genotyped for the mutations A376 G.G202A,A542 T,G680T and T968 C using TaqMan single nucleotide polymorphism assays and polymerase chain reaction followed by restriction fragment length polymorphism.Results:The prevalence of the G6 PD deficiency was 9.5%,in the study population.It was significantly higher in men compared to women(14.23%vs 6.0%,P=0.049).The 202A/376 G G6PD Awas the only deficient variant detected.Plasmodium falciparum asymptomatic parasitemia was significantly higher among the C6PD-non—deficient persons compared to the G6PD-deficient(P<0.001).The asymptomatic parasitemia was also significantly higher among G(SPI) nondeficient compared to C6PD—heterozygous females(P<0.001).Conclusions:This study showed that the G6 PD A- variant associated with protection against asymptomatic malaria in Burkina Faso is probably the most common deficient variant.

Is there any role of glucose-6-phosphate dehydrogenase in obesity induced metabolic disorder

The present study was designed to explore the possible mechanism of obesity associated metabolic syndrome. 150 subjects (120 men and 30 women) in the age-group of 17 - 26 years were studied. Body Mass Index and Waist-to-Hip Ratio were taken as a measure of generalized obesity and abdominal adiposity. The serum concentration of glucose-6-phosphate dehydrogenase increased with increasing levels of Body Mass Index and was found to be significant in obese subjects (Body Mass Index ≥ 30.0 kg/m2) and more so in the obese subjects with abdominal adiposity (p = 0.002) as compared to normal-weight subjects. Karl Pearson coefficient of correlation revealed a significant positive correlation of glucose-6-phosphate dehydrogenase with Body Mass Index (r = 0.499;p < 0.001) and malondialdehyde (a biomarker of oxidative stress) (r = 0.736;p < 0.001) but inverse correlation with adiponectin (r = -0.524;p < 0.001). Thus, we conclude that increased expression of glucose-6-phosphate dehydrogenase in obese subjects (more if it is associated with abdominal adiposity) might mediate the onset of obesity associated metabolic disorders by increasing oxidative stress.

Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency

Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics,often posing challenges for precise diagnoses using conventional methods.To this end,this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.Methods:The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis.Novel compound heterozygous variants of the GPI gene,c.174C>A(p.Asn58Lys)and c.1538G>T(p.Trp513Leu),were identified using whole-exome and Sanger sequencing.The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.Results:By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study,we found that most variants were located in exons 3,4,12,and 18,with a few localized in exons 8,9,and 14.This study identified novel compound heterozygous variants associated with GPI deficiency.These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.Conclusion:Early family-based sequencing analyses,especially for patients with congenital anemia,can help increase diagnostic accuracy for GPI deficiency,improve child healthcare,and enable genetic counseling.

Alleviation of PEGylated Puerarin on Erythrocyte Hemolysis Induced by Puerarin in Glucose-6-phosphate Dehydrogenase-deficient Rats

Objective To explore and analyze the reducing hemolytic effects of PEGylated puerarin (PEG-PUE) on erythrocytes induced by PUE in glucose-6-phosphate dehydrogenase (G6PD)-deficient rats. Methods The rat model with G6PD-deficiency was established via sc injecting 1% acetylphenyl-hydrazine. Then the G6PD-deficient erythrocyte suspension obtained from this rat model was used to evaluate the hemolytic effects of PUE and the reducing hemolytic effects of PEG-PUE via hemolytic activity and erythrocyte osmotic fragility assay. Results It was found that PUE could cause a serious hemolysis to the erythrocyte suspension with the increase of drug concentration and the prolongation of drug incubation time, the hemolytic rate of PUE was up to 40%, while the addition of PEG-PUE to the erythrocyte suspension revealed no significant hemolysis. Additionally, the result of erythrocyte osmotic fragility indicated that PEG-PUE exerted a slight effect on the erythrocyte membranes, and the NaCl concentration that induced 50% hemolysis (32 mmol/L) was about one-third PUE. Conclusion These results demonstrate that PEG-PUE could play a significant role in reducing the side effect of hemolysis induced by PUE. The low hemolytic activity of PEG-PUE makes it a favorable candidate for in vivo tests and PEG-PUE could also provide the useful insight for the further formulation development as an innovative drug.

The fungal endophyte Epichloëgansuensis increases NaCltolerance in Achnatherum inebrians through enhancing the activity of plasma membrane H^(+)-ATPase and glucose-6-phosphate dehydrogenase

Salt stress negatively affects plant growth,and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species,Achnatherum inebrians,to abiotic stresses.In this work,we first evaluated the effects of E.gansuensis on glucose-6-phosphate dehydrogenase(G6PDH)and plasma membrane(PM)H^(+)-ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations.Our results showed that the presence of E.gansuensis increased G6PDH,PMH^(+)-ATPase,superoxide dismutase and catalase activity to decrease O2•^(–),H_(2)O_(2)and Na^(+)contents in A.inebrians under NaCl stress,resulting in enhanced salt tolerance.In addition,the PM NADPH oxidase activity and NADPH/NADP+ratios were all lower in A.inebrians with E.ganusensis plants than A.inebrians plants without this endophyte under NaCl stress.In conclusion,E.gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H^(+)-ATPase to decrease ROS content.This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2―13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen inde-pendent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.

A diagnostic kit to screen individuals with glucose-6-phosphate dehydrogenase defect and its application on anti-malaria spot in the countryside

Objective To prepare a kit for screening individuals with glucose 6 phosphate dehydrogenase (G6PD) defect. The kit is easy to use and to get the fast as well as reliable results. Especially it is suitable for the anti malaria spots usually located in the remote countryside where no electricity is available. Methods The double filter paper method and other 2 techniques, the quantitative method and the single filter paper method, were used to determine G6PD activity in 70 samples of human erythrocytes. It was found that the results of the double filter paper method and those of the single filter paper method in the first 8 hours after the drying of the blood soaked filter paper were consistent with those of the quantitative method. When a piece of blood soaked paper is left under room temperature more than 24 hours, G6PD in the erythrocytes deteriorated spontaneously and consequently the number of positive cases increased along with the elapse of time.Results Satisfactory results were achieved when the kit was used to screen cases of G6PD defect from 151 farmers who were receiving anti mararia therapy. The kit was made according to a technique named “double filter paper” method.Conclusions These findings suggest that the double filter paper method can reveal the level of G6PD activity and the results are rapidly obtained when the method is used on the anti malaria spot.

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis

Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

Separation and Purification of GST-glycerol-3-phosphate Dehydrogenase

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.

HEXOKINASE, GLUCOSE-6-PHOSPHATASE DEHYDROGENASE AND ALEOSE REDUCTASE IN HUMAN FETAL LENSES

The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a negetive correlation between the age of fetus and activity of AR(r=-0.810 1,0.05>P>0.01).

Structure and Expression Analyses of a Gene Encoding Fructose-6-Phosphate, 2-Kinase/Fructose-2,6-Bisphosphatase from Maize

A full-length cDNA encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from maize (Zea mays L.) was cloned by the methods of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and designated as mF2KP. The encoded protein is composed of two regions. Its COOH-terminal region is catalytic region and homologous to the enzymes from other eukaryotes; and its NH 2-terminal region is common and special region only in plant. A truncated fragment of mF2KP covering integrated catalytic region was expressed in Escherichia coli. The fusion protein had the activities of fructose-6-phosphate, 2-kinase as well as fructose-2,6-bisphosphatase. Northern blot showed that the transcript level of mF2KP in seedlings initiated from strong-vigor seeds is lower than that from weak-vigor seeds.