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1.
Arch Insect Biochem Physiol ; 102(4): e21618, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31512274

RESUMO

The freeze-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose-6-phosphate dehydrogenase (G6PDH) mediates the rate-limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (-15°C) E. solidaginis larvae by a single-step chromatography method utilizing 2',5'-ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in Km for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.


Assuntos
Congelamento , Glucosefosfato Desidrogenase/metabolismo , Tephritidae/metabolismo , Animais , Crioprotetores , Glucosefosfato Desidrogenase/genética , Larva/enzimologia , Larva/genética , Larva/metabolismo , Via de Pentose Fosfato , Tephritidae/enzimologia , Tephritidae/genética , Tephritidae/crescimento & desenvolvimento , Ubiquitinação
2.
Microb Cell Fact ; 18(1): 161, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547820

RESUMO

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Ácidos Graxos/metabolismo , Glucosefosfato Desidrogenase/genética , Dióxido de Carbono/análise , Diatomáceas/metabolismo , Engenharia Genética , Glucosefosfato Desidrogenase/metabolismo , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
4.
Malar J ; 18(1): 209, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234865

RESUMO

BACKGROUND: To reduce onward falciparum malaria transmission, the World Health Organization recommends adding single low-dose (SLD) primaquine to artemisinin-based combination treatment in low transmission areas. However, uptake of this recommendation has been relatively slow given concerns about whether individual risks justify potential community benefit. This study was undertaken to generate comprehensive local data on the risk-benefit profile of SLD primaquine deployment in a pre-elimination area in South Africa. METHODS: This randomized, controlled open-label trial investigated adding a single low primaquine dose on day 3 to standard artemether-lumefantrine treatment for uncomplicated falciparum malaria. Efficacy, safety and tolerability of artemether-lumefantrine and primaquine treatment were assessed on days 3, 7, 14, 28 and 42. Lumefantrine concentrations were assayed from dried blood spot samples collected on day 7. RESULTS: Of 217 patients screened, 166 were enrolled with 140 randomized on day 3, 70 to each study arm (primaquine and no primaquine). No gametocytes were detected by either microscopy or PCR in any of the follow-up samples collected after randomization on day 3, precluding assessment of primaquine efficacy. Prevalence of the CYP2D6*4, CYP2D6*10 and CYP2D6*17 mutant alleles was low with allelic frequencies of 0.02, 0.11 and 0.16, respectively; none had the CYP2D6*4/*4 variant associated with null activity. Among 172 RDT-positive patients G6PD-genotyped, 24 (14%) carried the G6PD deficient (A-) variant. Median haemoglobin concentrations were similar between treatment arms throughout follow-up. A third of participants had a haemoglobin drop > 2 g/dL; this was not associated with primaquine treatment but may be associated with G6PD genotype [52.9% (9/17) with A- genotype vs. 31% (36/116) with other genotypes (p = 0.075)]. Day 7 lumefantrine concentrations and the number and nature of adverse events were similar between study arms; only one serious adverse event occurred (renal impairment in the no primaquine arm). The artemether-lumefantrine PCR-corrected adequate clinical and parasitological response rate was 100%, with only one re-infection found among the 128 patients who completed 42-day follow-up. CONCLUSIONS: Safety, tolerability, CYP2D6 and G6PD variant data from this study support the deployment of the WHO-recommended SLD primaquine without G6PD testing to advance malaria elimination in South African districts with low-intensity residual transmission. Trial registration Pan African Clinical Trial Registry, PACTR201611001859416. Registered 11 November 2016, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1859.


Assuntos
Antimaláricos/uso terapêutico , Malária Falciparum/tratamento farmacológico , Primaquina/uso terapêutico , Adulto , Antimaláricos/efeitos adversos , Combinação Arteméter e Lumefantrina/uso terapêutico , Citocromo P-450 CYP2D6/genética , Feminino , Frequência do Gene , Genótipo , Glucosefosfato Desidrogenase/genética , Humanos , Lumefantrina/sangue , Masculino , Mutação , Primaquina/efeitos adversos , África do Sul , Resultado do Tratamento
5.
Niger Postgrad Med J ; 26(2): 118-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31187752

RESUMO

Background: Sickle cell disease (SCD) and glucose-6-phosphate dehydrogenase (G6PD) deficiency are inherited disorders associated with chronic haemolysis. Therefore, coinheritance of both disorders could worsen haemolysis in the former and compound a haemolytic crisis. This study compared clinical and laboratory features of deficient and non-deficient SCD patients and the G6PD activities of SCD patients and apparently healthy controls. Materials and Methods: This is a case-control study of 175 SCD patients and 166 non-SCD controls. G6PD assay was carried out on haemolysate from washed red cells. The G6PD activity was measured by spectrophotometry. Results: The mean age of patients and controls was 27.3 ± 9.4 and 35.9 ± 9.7 years, respectively, with 75 (46.2%) and 87 (52.4%) being males, respectively. G6PD activity was similar in cases and controls (6.7 ± 3.3 vs. 6.9 ± 3.0 IU/gHb), respectively (P = 0.6). The prevalence of G6PD deficiency was higher in patients than controls (28.6% vs. 22.3%, P = 0.18), and SCD patients were twice more likely to have enzyme activities below 3.0 IU/gHb. No significant difference was observed in the clinical parameters between deficient and non-deficient patients. Deficient patients were more likely to have lower haematocrit (22.8 ± 3.9% vs. 24.5 ± 5%, P = 0.04) and non-significantly higher bilirubin and reticulocyte counts. Furthermore, in patients, severe deficiency resulted in higher bilirubin than in those with mild deficiency (60.5 vs. 21.7 IU/L, P < 0.001). G6PD activity correlated positively with haematocrit (r = 0.91, P = 0.01) and mean corpuscular haemoglobin concentration (r = 0.17, P = 0.02). Conclusions: Coinheritance of both disorders could worsen haemolysis in SCD patients, and care should, therefore, be taken in the choice of drugs in deficient SCD patients.


Assuntos
Anemia Falciforme/complicações , Anemia Falciforme/genética , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Hemólise/genética , Adolescente , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/epidemiologia , Bilirrubina/sangue , Estudos de Casos e Controles , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/sangue , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Hemoglobinas/análise , Humanos , Masculino , Nigéria/epidemiologia , Prevalência , Adulto Jovem
6.
Int J Med Sci ; 16(5): 623-629, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217728

RESUMO

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in Western Countries. Evidence indicates that Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency, a common genetic abnormality, may protect against ischemic heart and cerebrovascular disease, ocular vascular disorders, and colorectal cancer. This study was undertaken to ascertain whether G6PD deficiency may protect against AMD. Materials and Methods: 79 men with late-stage AMD and 79 male, age-matched cataract controls without AMD were recruited in March-December 2016. Smoking status, clinical history, and drug use were recorded. A blood sample was taken from each participant. Complete blood count, hemoglobin, glucose, creatinine, cholesterol, triglycerides, transaminases, bilirubin, and erythrocyte G6PD activity were measured. Stepwise logistic regression was used to investigate the association between G6PD deficiency and AMD. Results: G6PD deficiency was found in 7 (8.9%) AMD patients and 8 (10.1%) controls, a not statistically significant difference. Stepwise logistic regression disclosed that AMD was significantly associated with increased diastolic blood pressure (OR=1.09, 95% CI=1.03-1.15, P=0.02) and LDL-cholesterol (OR=1.02, 95% CI=1.0001-1.03, P=0.049) and lower values of white blood cell (WBC) count (OR=0.71, 95% CI=0.56-0.88, P=0.02) and aspartate aminotransferase (AST) (OR=0.92, 95% CI=0.85-0.99, P=0.044). Conclusion: Results suggest that G6PD deficiency has no protective effect on nor is a risk factor for AMD. Larger studies are necessary to confirm whether increased diastolic blood pressure and LDL-cholesterol and lower values of WBC count and AST are risk factors for AMD.


Assuntos
Catarata/metabolismo , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Degeneração Macular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Catarata/epidemiologia , Catarata/genética , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Humanos , Itália/epidemiologia , Degeneração Macular/epidemiologia , Degeneração Macular/genética , Masculino , Pessoa de Meia-Idade , Fatores de Risco
7.
Biotechnol Appl Biochem ; 66(4): 591-596, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31050059

RESUMO

Glucose-6-phosphate dehydrogenase (G6PDH) has been used in enzyme multiplied immunoassay technique (EMIT) assays for detecting small molecule metabolites such as cholyglycine (CG). A key parameter for successful EMIT CG assay development is the inhibition rate of the G6PDH-CG conjugate, measured as the decrease in enzyme activity upon CG antibody binding. Several commonly used G6PDH cysteine mutants including A45C and K55C have been labeled with CG-maleimide derivative, but inhibition rates of are unsatisfactory. Herein, we investigated whether other mutation sites can achieve better inhibition rates. We generated eight cysteine mutants (K106C, Y155C, A201C, T258C, D306C, D375C, G426C, and D480C) of G6PDH, measured their inhibition rates, and evaluated the performance of the D306C mutant using EMIT CG assays. One of the eight mutants (D306C) displayed improved inhibition rate, whereas all others exhibited inhibition similar to or lower than that of A45C and K55C. The enhanced inhibition rate of D306C improved the EMIT CG assay calibration curve, using an Abbott c16000 automated biochemical analyzer, resulting in better repeatability, precision, and linearity than with K55C assays and a commercially available EMIT CG kit. The G6PDH mutant D306C has a higher inhibition rate in EMIT CG assays and improves assay performance.


Assuntos
Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Ácido Glicocólico/análise , Imunoensaio , Mutação , Bibliotecas de Moléculas Pequenas/análise , Cisteína/genética , Glucosefosfato Desidrogenase/química , Ácido Glicocólico/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/metabolismo
8.
Gene ; 707: 143-150, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31075411

RESUMO

OBJECTIVE: Present study was undertaken to study the association between sickle cell anemia (SCA) and glucose-6-phosphate dehydrogenase (G6PD) deficiency from Sahu and Kurmi population of Durg and Rajnandgaon district of Chhattisgarh, India. METHOD: A random sampling of 1749 individuals was done. SCA and G6PD deficiency was detected by slide test followed by electrophoresis and Enzymatic reaction indicated by change in colour respectively. Further the samples were subjected to analyze glutathione-S-transferase (GST) i.e. GSTM1 and GSTT1 gene polymorphism, variance of G6PD among G6PD deficient samples by PCR-RFLP. Oxidative stress and DNA damage by comet assay was also analyzed. RESULTS: Present finding indicates positive correlation between SCA and G6PD deficiency in Durg and Rajnandgaon district [Durg: (r = 0.92; HbAS-G6PDd and r = 0.56; HbSS-G6PDd) Rajnandgaon: (r = 0.63; HbAS-G6PDd and r = 0.86; HbSS-G6PDd)]. Significant changes (P < 0.05) in antioxidant enzymatic parameters were observed in HbSS and G6PD with sickle positive individual. Assessment of DNA damage by Comet assay considering Head DNA percent, Tail DNA percent, Tail length and Tail moment also showed significant changes (P < 0.05) within all concerned parameters in HbSS and G6PD with sickle positive individual. Analysis of GST gene polymorphism showed that frequency of individuals carrying the GSTM1 null genotype was higher in HbAS (60%) and the frequency of individual carrying the GSTT1 null genotype was found higher in HbSS (66.6%). G6PD variants analysis also confirmed the presence of highest percentage of mutation among G6PD deficient population as compared to control and a positive correlation was observed between G6PD deficiency and mutant variants of G6PD gene [Rajnandgaon: (r = 0.67; G6PDd-Mahidol mutated and r = 0.90; G6PDd-Union mutated) Durg: (r = 0.91; G6PDd-Mahidol mutated and r = 0.01; G6PDd-Union mutated)] . CONCLUSION: Thus present finding indicates positive correlation between SCA and G6PD deficiency in Chhattisgarh, India.


Assuntos
Anemia Falciforme/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Glucosefosfato Desidrogenase/genética , Glutationa Transferase/genética , Anemia Falciforme/genética , Dano ao DNA , Feminino , Genótipo , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Índia/epidemiologia , Masculino , Mutação , Estresse Oxidativo , Polimorfismo de Fragmento de Restrição , Traço Falciforme/epidemiologia , Traço Falciforme/genética
9.
Appl Microbiol Biotechnol ; 103(14): 5781-5796, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139900

RESUMO

Inhibitory compounds liberated from lignocellulose pretreatment are representative toxic chemicals that repress microbial growth and metabolism. A tolerant strain of the industrial yeast Saccharomyces cerevisiae is able to detoxify a major class of toxic compounds while producing ethanol. Knowledge on the yeast tolerance was mostly obtained by gene expression analysis and limited protein expression evidence is yet available underlying the yeast adaptation. Here we report a comparative protein expression profiling study on Y-50049, a tolerant strain compared with its parental industrial type strain Y-12632. We found a distinctive protein expression of glucose-6-phosphate dehydrogenase (Zwf1) in Y-50049 but not in Y-12632, in the relatively conserved glycolysis and pentose phosphate pathway (PPP) in response to a combinational challenge of 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF). A group of proteins with aldehyde reduction activity was uniquely induced expressed in Y-50049 but not in Y-12632. Such evidence allowed fine-tuning a mechanism of the renovated in situ detoxification by Y-50049. As the key protein, Zwf1 drove the glucose metabolism in favor of the oxidative branch of the PPP facilitating in situ detoxification of the toxic chemicals by Y-50049. The activated expression of Zwf1 generated the essential cofactor nicotinamide adenine dinucleotide phosphate (NADPH) enabling reduction of furfural and HMF through a group of aldehyde reduction enzymes. In return, the activate aldehyde reductions released desirable feedbacks of NADP+ stimulating continued oxidative activity of Zwf1. Thus, a well-maintained cofactor regeneration cycle was established to restore the cofactor imbalance caused by furfural-HMF. Challenges and perspectives on adaptation of significantly differential expressions of ribosomal proteins and other unique proteins are also discussed.


Assuntos
Etanol/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Aldeídos/metabolismo , Regulação Fúngica da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glicólise , Inativação Metabólica , Microbiologia Industrial , NADP/metabolismo , Via de Pentose Fosfato , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
10.
J Exp Clin Cancer Res ; 38(1): 160, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30987650

RESUMO

BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment. METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers. RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients. CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Lapatinib/farmacologia , Prognóstico , Recidiva
11.
Toxicol Lett ; 310: 23-30, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30980912

RESUMO

The aim of this study was to determine whether Pb affects glucose metabolism in the hippocampus of rats. Male Sprague-Dawley rats aged 21 days were orally administered a 0.1%, 0.2%, or 0.3% lead acetate solution in deionized water for 65 days. Then, the weight of the rats; brain Pb content; brain glucose levels; activities of hexokinase, fructose-6-phosphate kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase; expression of genes related to the insulin signaling pathway; as well as the gene and protein expression of glucose transporter (GLUT)-1 and GLUT-3 in the hippocampus were evaluated. The results showed that Pb content in the brain tissue of rats in the dose groups significantly increased, whereas the body weight gain, activities of glucose metabolism-related enzymes, and expression of the insulin signaling pathway-related genes significantly decreased compared to the corresponding values in the control group. In comparison with the control group, the brain glucose levels increased significantly in the low-dose group, but there were no significant differences with the middle- and high-dose groups. Furthermore, the mRNA of GLUT-1 in the three dose groups and the GLUT-3 in the middle- and high-dose groups rose markedly, while the GLUT-1 and GLUT-3 protein expression significantly increased in the middle- and high-dose groups and in the high-dose group, respectively. Taken together, the results showed that Pb exposure resulted in a lower body weight gain, higher brain Pb content and also affected brain glucose metabolism and the insulin signaling pathway.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Hipocampo/efeitos dos fármacos , Insulina/metabolismo , Compostos Organometálicos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos Sprague-Dawley , Medição de Risco , Ganho de Peso/efeitos dos fármacos
12.
Food Chem ; 289: 112-120, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955592

RESUMO

Sucrose acts as a vital signal that modulates fruit ripening. In current study, 50 mM sucrose was applied in strawberry fruit to investigate the regulation of sucrose in anthocyanin synthesis after harvest. The results showed that sucrose treatment increased the contents of glucose, fructose and sucrose, which were 19.76%, 15.83% and 16.50% higher, respectively, compared with control at the end of storage. The increase of glucose and fructose contents resulted from the activation of acid invertase by sucrose treatment. In addition, sucrose treatment specifically increased four pelargonidin derivatives, pelargonidin 3-glucoside, pelargonidin 3-rutinoside, pelargonidin 3-malonylglucoside and pelargonidin 3-methylmalonyglucoside, during the storage. Further, transcriptional profiles and enzyme activities analysis revealed that the accumulation of pelargonidin derivatives was related to the activation of the pentose phosphate pathway, shikimate pathway, phenylpropanoid pathway, and flavonoid pathway. These results provided new insights into the regulation of sucrose on the accumulation of individual anthocyanins.


Assuntos
Antocianinas/biossíntese , Fragaria/metabolismo , Antocianinas/análise , Antocianinas/metabolismo , Flavonoides/metabolismo , Fragaria/química , Frutas/química , Frutas/metabolismo , Glucose/análise , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Espectrometria de Massas , Via de Pentose Fosfato/efeitos dos fármacos , Fenilalanina Amônia-Liase/metabolismo , Sacarose/farmacologia , Transcinamato 4-Mono-Oxigenase/metabolismo
13.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889888

RESUMO

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5⁻8.5 and 30 °C for NADP⁺ and 20 °C for NAD⁺. SpG6PD1 utilized both NADP⁺ and NAD⁺, with the preferential utilization of NADP⁺. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4⁻40 °C, its activity and tertiary structure were better preserved between 4⁻20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.


Assuntos
Glucosefosfato Desidrogenase/genética , Sphingomonas/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática/efeitos dos fármacos , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Concentração de Íons de Hidrogênio , Íons , Cinética , Metais/farmacologia , Análise Espectral , Temperatura Ambiente , Termodinâmica
14.
Mol Pain ; 15: 1744806919838659, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838902

RESUMO

BACKGROUND AND AIM: Diabetic neuropathic pain is a refractory and disabling complication of diabetes mellitus. The pathogenesis of the diabetic neuropathic pain is still unclear, and treatment is insufficient. The aim of this study is to investigate the roles of glucose-6-phosphate dehydrogenase (G6PD) and toll-like receptor 4 (TLR4) in neuropathic pain in rats with diabetes. METHODS: Type 1 diabetes model was induced by intraperitoneal injection of streptozotocin (STZ, 75 mg/kg) in adult female Sprague-Dawley rats. Paw withdrawal threshold and paw withdrawal latency of rats were measured by von Frey filaments and thermal radiation, respectively. The expressions of G6PD and TLR4 in L4-L6 dorsal root ganglions (DRGs) were measured by western blotting and quantitative real-time polymerase chain reaction analysis. Fluorescent immunohistochemistry was employed to detect expressions of G6PD and TLR4 and co-location of G6PD with TLR4. RESULTS: The mRNA and protein expression levels of G6PD in DRGs were significantly decreased in diabetic rats when compared with age-matched control rats. Upregulation of G6PD by intrathecal injection of G6PD overexpression adenovirus markedly attenuated hindpaw pain hypersensitivity of diabetic rats. The mRNA and protein expression levels of TLR4 in DRGs of diabetic rats were significantly increased when compared with control rats. Intrathecal injection of TLR4-selective inhibitor CLI-095 attenuated diabetic pain in dose- and time-dependent manners. Furthermore, G6PD and TLR4 were co-localized in DRG neurons. Intrathecal injection of G6PD overexpression adenovirus greatly reduced TLR4 expression, while intrathecal injection of CLI-095 had no significant effect on G6PD expression in diabetic rats. CONCLUSIONS: Our results suggest that decrease in G6PD expression was involved in diabetic peripheral neuropathic pain, which was most likely through upregulation of TLR4 expression in the DRGs of rats.


Assuntos
Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Glucosefosfato Desidrogenase/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética
15.
Malar J ; 18(1): 75, 2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866940

RESUMO

BACKGROUND: Primaquine is effective against the latent liver stage of Plasmodium vivax. Eliminating the latent liver stage of P. vivax is one of the necessary conditions to achieve the goal of malaria elimination in Lao People's Democratic Republic (PDR) by 2030. However, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk of haemolysis when ingesting primaquine. The aim of this study was to detect the prevalence of the G6PD Viangchan variant, which is said to be common in Lao PDR and which can result in severe haemolysis in patients exposed to primaquine. METHODS: Blood samples were collected from villagers in three malaria endemic provinces: Champasak and Savannakhet in the south, and Phongsaly in the north. Each blood sample was semi-quantitatively assayed for G6PD enzyme activity using the G6PD Assay Kit-WST Lyophilized (DOJINDO Laboratories, Japan). Blood samples that were found to be G6PD deficient were sequenced to detect G6PD Viangchan mutation. RESULTS: In total, 2043 blood samples were collected from Phongsaly (n = 426, 20.9%), Savannakhet (n = 924, 45.2%), and Champasak (n = 693, 33.9%) provinces in Lao PDR from 2016 to 2017. Of these, 964 (47.2%) were taken from male villagers and 1079 (52.8%) were taken from female villagers. G6PD Viangchan mutation was not detected in Phongsaly province in this study. In Savannakhet province, 48 of the 924 samples (45 males, 3 females) had the G6PD Viangchan mutation (n = 48, 5.2%). In Champasak province, 42 of the 693 samples (18 males, 24 females) had the G6PD Viangchan mutation (n = 42, 6.1%). CONCLUSIONS: G6PD Viangchan variant, which can cause severe haemolysis in the carrier when exposed to primaquine, was detected among 6.1% of the villagers in Champasak and 5.2% in Savannakhet but not in Phongsaly in this study. G6PD Viangchan variant might be common in the south of Laos but not so in the north. In the north, other G6PD deficiency variants might be more prevalent. However, in order not to overlook anyone and ensure a safe primaquine therapy for people living in malaria endemic areas in Lao PDR, G6PD testing is necessary.


Assuntos
Erradicação de Doenças/métodos , Genótipo , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Glucosefosfato Desidrogenase/genética , Malária Vivax/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Glucosefosfato Desidrogenase/análise , Hemólise , Humanos , Lactente , Recém-Nascido , Laos/epidemiologia , Malária Vivax/prevenção & controle , Masculino , Pessoa de Meia-Idade , Prevalência , Primaquina/efeitos adversos , População Rural , Análise de Sequência de DNA , Adulto Jovem
16.
J Microbiol Biotechnol ; 29(4): 577-586, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30786701

RESUMO

The engineered Aspergillus oryzae has a high NADPH demand for the xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, which is the main enzyme responding for the NADPH regeneration. The open reading frame and cDNA of putative A. oryzae G6PDH (AoG6PDH) were obtained, which was followed by heterogeneous expression in Escherichia coli and was purified as a his6-tagged protein presently. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed the maximal activity at pH 7.5 and the optimal temperature was 50 °C. This enzyme had half-life time of 33.3 min at 40 °C. Kinetics assay showed that AoG6PDH was strictly dependent on NADP+ (Km = 6.3 µM, kcat = 1000.0 s-1, kcat/Km =158.7 s-1·µM-1) as cofactor. The Km and kcat/Km value of glucose-6-phosphate were 109.7 s-1·µM-1 and 9.1 -1·µM-1 respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate steady-state ordered BiBi mechanism, where NADP+ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae for improving the xylose utilization and yields of valued metabolites.


Assuntos
Aspergillus oryzae/metabolismo , Glucosefosfato Desidrogenase/biossíntese , Engenharia Metabólica/métodos , Via de Pentose Fosfato , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Clonagem Molecular , DNA Fúngico , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , NADP/metabolismo , Oxirredução , Via de Pentose Fosfato/genética , Temperatura Ambiente , Xilose/metabolismo
17.
Malar J ; 18(1): 22, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683097

RESUMO

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.


Assuntos
Hidrolases de Éster Carboxílico/genética , Glucosefosfato Desidrogenase/genética , Malária Vivax/genética , Complexos Multienzimáticos/genética , Proteínas de Protozoários/genética , Hidrolases de Éster Carboxílico/metabolismo , Clonagem Molecular , Citosol/metabolismo , Escherichia coli/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Cinética , Malária Vivax/enzimologia , Malária Vivax/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredução , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Malar J ; 18(1): 14, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665411

RESUMO

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest. METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods. RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype. CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.


Assuntos
Anemia Falciforme/diagnóstico , Técnicas de Genotipagem/métodos , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Doença da Hemoglobina C/diagnóstico , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Burkina Faso , Criança , Glucosefosfato Desidrogenase/genética , Hemoglobina C/genética , Hemoglobina Falciforme/genética , Humanos , Malária/complicações , Masculino , Pessoa de Meia-Idade , Adulto Jovem
20.
PLoS One ; 14(1): e0209204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30601843

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD) deficiency may affect the clinical presentation of dengue due to the altered redox state in immune cells. We aimed to determine the association between G6PD deficiency and severity of dengue infection in paediatric patients in Myanmar. A cross-sectional study was conducted among paediatric patients aged 2-13 years with dengue in Yankin Children Hospital, Myanmar. One hundred and ninety-six patients positive for dengue infection, as determined via PCR or ELISA, were enrolled. Dengue severity was determined according to the 2009 WHO classification guidelines. Spectrophotometric assays determined G6PD levels. The adjusted median G6PD value of males in the study population was used to define various cut-off points according to the WHO classification guidelines. G6PD genotyping for Mahidol, Kaiping and Mediterranean mutations was performed for 128 out of 196 samples by real-time multiplex PCR. 51 of 196 (26.0%) patients had severe dengue. The prevalence of G6PD phenotype deficiency (< 60% activity) in paediatric patients was 14.8% (29/196), specifically, 13.6% (14/103) in males and 16.2% (15/93) in females. Severe deficiency (< 10% activity) accounted for 7.1% (14/196) of our cohort, occurring 11.7% (12/103) in males and 2.2% (2/93) in females. Among 128 samples genotyped, the G6PD gene mutations were detected in 19.5% (25/128) of patients, with 20.3% (13/ 64) in males and 18.8% (12/64) in females. The G6PD Mahidol mutation was 96.0% (24/25) while the G6PD Kaiping mutation was 4.0% (1/25). Severe dengue was not associated with G6PD enzyme deficiency or presence of the G6PD gene mutation. Thus, no association between G6PD deficiency and dengue severity could be detected. Trial registration: The study was registered following the WHO International Clinical Trials Registry Platform (WHO-ICTRP) on Thai Clinical Trials Registry (TCTR) website, registration number # TCTR20180720001.


Assuntos
Dengue/complicações , Dengue/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Feminino , Genótipo , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/enzimologia , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Mutação , Mianmar/epidemiologia , Prevalência , Estudos Prospectivos
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