RESUMEN

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.

Asunto(s)

Escherichia coli , Glucosafosfato Deshidrogenasa , NADP , Oxidación-Reducción , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa , Glucosafosfato Deshidrogenasa/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , NADP/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Ribulosafosfatos/metabolismo , Glucosa-6-Fosfato/metabolismo , Peróxidos/metabolismo , Hidrolasas de Éster Carboxílico

RESUMEN

The enzyme glucose-6-phosphate dehydrogenase (G6PDH) plays crucial roles in glucose homeostasis and the pentose phosphate pathway (PPP), being also involved in redox metabolism. The PPP is an important metabolic pathway that produces ribose and nicotinamide adenine dinucleotide phosphate (NADPH), which are essential for several physiologic and biochemical processes, such as the synthesis of fatty acids and nucleic acids. As a rate-limiting step in PPP, G6PDH is a highly conserved enzyme and its deficiency can lead to severe consequences for the organism, in particular for cell growth. Insufficient G6PDH activity can lead to cell growth arrest, impaired embryonic development, as well as a reduction in insulin sensitivity, inflammation, diabetes, and hypertension. While research on G6PDH and PPP has historically focused on mammalian models, particularly human disorders, recent studies have shed light on the regulation of this enzyme in arthropods, where new functions were discovered. This review will discuss the role of arthropod G6PDH in regulating redox homeostasis and immunometabolism and explore potential avenues for further research on this enzyme in various metabolic adaptations.

RESUMEN

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd) is an X-linked disorder affecting over 400 million people worldwide. Individuals with molecular variants associated with reduced enzymatic activity are susceptible to oxidative stress in red blood cells, thereby increasing the risk of pathophysiological conditions and toxicity to anti-malarial treatments. Globally, the prevalence of G6PDd varies among populations. Accordingly, this study aims to characterize G6PDd distribution within the Ecuadorian population and to describe the spatial distribution of reported malaria cases. METHODS: Molecular variants associated with G6PDd were genotyped in 581 individuals from Afro-Ecuadorian, Indigenous, Mestizo, and Montubio ethnic groups. Additionally, spatial analysis was conducted to identify significant malaria clusters with high incidence rates across Ecuador, using data collected from 2010 to 2021. RESULTS: The A- c.202G > A and A- c.968T > C variants underpin the genetic basis of G6PDd in the studied population. The overall prevalence of G6PDd was 4.6% in the entire population. However, this frequency increased to 19.2% among Afro-Ecuadorian people. Spatial analysis revealed 12 malaria clusters, primarily located in the north of the country and its Amazon region, with relative risks of infection of 2.02 to 87.88. CONCLUSIONS: The findings of this study hold significant implications for public health interventions, treatment strategies, and targeted efforts to mitigate the burden of malaria in Ecuador. The high prevalence of G6PDd among Afro-Ecuadorian groups in the northern endemic areas necessitates the development of comprehensive malaria eradication strategies tailored to this geographical region.

Asunto(s)

Deficiencia de Glucosafosfato Deshidrogenasa , Malaria , Humanos , Ecuador/epidemiología , Eritrocitos , Etnicidad , Deficiencia de Glucosafosfato Deshidrogenasa/epidemiología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Malaria/epidemiología

RESUMEN

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes.

Asunto(s)

Anemia Hemolítica Congénita no Esferocítica , Anemia de Células Falciformes , Humanos , Antioxidantes , Eritrocitos , Estrés Oxidativo

RESUMEN

AIMS: To report the case of chronic osteomyelitis of a maxilla in a woman with uncontrolled diabetes mellitus (DM), glucose-6-phosphate dehydrogenase (G6PD) deficiency and mental illness, in an attempt to clarify its pathogenesis and treatment. METHODS AND RESULTS: A case of a woman with moderate G6PD deficiency (Class III) who developed bilateral and asynchronous chronic suppurative osteomyelitis (CSO) of her maxilla with extensive bone sequestra, fistulae and whose management was performed by local surgery for bony sequestra and fistulae removal; closure communication under 4 weeks antibiotic cover. CONCLUSIONS: CSO of the jaw may be a complication of the G6PD deficiency and DM and its severity depends on patient's medical status.

Asunto(s)

Enfermedades Óseas , Diabetes Mellitus , Deficiencia de Glucosafosfato Deshidrogenasa , Osteomielitis , Femenino , Humanos , Maxilar , Osteomielitis/complicaciones , Osteomielitis/cirugía

RESUMEN

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 µM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.

Asunto(s)

Aminoácidos , Leuconostoc mesenteroides , Aminoácidos/química , Glucosafosfato Deshidrogenasa/química , Oxidantes/química , Oxidación-Reducción , Peróxidos , Ácido Peroxinitroso , Desplegamiento Proteico

RESUMEN

Glucose-6-phosphate dehydrogenase (G6PD) is the second rate-limiting enzyme of the pentose phosphate pathway. This enzyme is present in the cytoplasm of all mammalian cells, and its activity is essential for an adequate functioning of the antioxidant system and for the response of innate immunity. It is responsible for the production of nicotinamide adenine dinucleotide phosphate (NADPH), the first redox equivalent, in the pentose phosphate pathway. Viral infections such as SARS-CoV-2 may induce the Warburg effect with an increase in anaerobic glycolysis and production of lactate. This condition ensures the success of viral replication and production of the virion. Therefore, the activity of G6PD may be increased in COVID-19 patients raising the level of the NADPH, which is needed for the enzymatic and non-enzymatic antioxidant systems that counteract the oxidative stress caused by the cytokine storm. G6PD deficiency affects approximately 350-400 million people worldwide; therefore, it is one of the most prevalent diseases related to enzymatic deficiency worldwide. In G6PD-deficient patients exposed to SARS-CoV-2, the amount of NADPH is reduced, increasing the susceptibility for viral infection. There is loss of the redox homeostasis in them, resulting in severe pneumonia and fatal outcomes.

Asunto(s)

COVID-19 , Glucosafosfato Deshidrogenasa , Animales , Antioxidantes , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Mamíferos/metabolismo , NADP/metabolismo , SARS-CoV-2

RESUMEN

Helicobacter pylori (H. pylori) has been proposed as the foremost risk factor for the development of gastric cancer. We found that H. pylori express the enzyme glucose-6-phosphate dehydrogenase (HpG6PD), which participates in glucose metabolism via the pentose phosphate pathway. Thus, we hypothesized that if the biochemical and physicochemical characteristics of HpG6PD contrast with the host G6PD (human G6PD, HsG6PD), HpG6PD becomes a potential target for novel drugs against H. pylori. In this work, we characterized the biochemical properties of the HpG6PD from the H.pylori strain 29CaP and expressed the active recombinant protein, to analyze its steady-state kinetics, thermostability, and biophysical aspects. In addition, we analyzed the HpG6PD in silico structural properties to compare them with those of the HsG6PD. The optimal pH for enzyme activity was 7.5, with a T1/2 of 46.6 °C, at an optimum stability temperature of 37 °C. The apparent Km values calculated for G6P and NADP+ were 75.0 and 12.8 µM, respectively. G6P does not protect HpG6PD from trypsin digestion, but NADP+ does protect the enzyme from trypsin and guanidine hydrochloride (Gdn-HCl). The biochemical characterization of HpG6PD contributes to knowledge regarding H. pylori metabolism and opens up the possibility of using this enzyme as a potential target for specific and efficient treatment against this pathogen; structural alignment indicates that the three-dimensional (3D) homodimer model of the G6PD protein from H. pylori is different from the 3D G6PD of Homo sapiens.

RESUMEN

Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the Halobacteria class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored. Here we report the structural and kinetic characteristics of glucose-6-phosphate dehydrogenase from Haloferax volcanii (HvG6PDH) belonging to the short-chain dehydrogenases/reductases (SDR) superfamily. The enzyme was expressed in Escherichia coli and successfully solubilized and refolded from inclusion bodies. The enzyme is active in the presence of several salts, though the maximum activity is achieved in the presence of KCl, mainly by an increment in the k cat value, that correlates with a diminution of its flexibility according to molecular dynamics simulations. The high K M for glucose-6-phosphate and its promiscuous activity for glucose restrict the use of HvG6PDH as an auxiliary enzyme for the determination of halophilic glucokinase activity. Phylogenetic analysis indicates that SDR-G6PDH enzymes are exclusively present in Halobacteria, with HvG6PDH being the only enzyme characterized. Homology modeling and molecular dynamics simulations of HvG6PDH identified a conserved NLTX2H motif involved in glucose-6-phosphate interaction at high salt concentrations, whose residues could be crucial for substrate specificity. Structural differences in its conformational dynamics, potentially related to the haloadaptation strategy, were also determined.

RESUMEN

Giardia lamblia is a single-celled eukaryotic parasite with a small genome and is considered an early divergent eukaryote. The pentose phosphate pathway (PPP) plays an essential role in the oxidative stress defense of the parasite and the production of ribose-5-phosphate. In this parasite, the glucose-6-phosphate dehydrogenase (G6PD) is fused with the 6-phosphogluconolactonase (6PGL) enzyme, generating the enzyme named G6PD::6PGL that catalyzes the first two steps of the PPP. Here, we report that the G6PD::6PGL is a bifunctional enzyme with two catalytically active sites. We performed the kinetic characterization of both domains in the fused G6PD::6PGL enzyme, as well as the individual cloned G6PD. The results suggest that the catalytic activity of G6PD and 6PGL domains in the G6PD::6PGL enzyme are more efficient than the individual proteins. Additionally, using enzymatic and mass spectrometry assays, we found that the final metabolites of the catalytic reaction of the G6PD::6PGL are 6-phosphoglucono-δ-lactone and 6-phosphogluconate. Finally, we propose the reaction mechanism in which the G6PD domain performs the catalysis, releasing 6-phosphoglucono-δ-lactone to the reaction medium. Then, this metabolite binds to the 6PGL domain catalyzing the hydrolysis reaction and generating 6-phosphogluconate. The structural difference between the G. lamblia fused enzyme G6PD::6PGL with the human G6PD indicate that the G6PD::6PGL is a potential drug target for the rational synthesis of novels anti-Giardia drugs.

RESUMEN

Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.

Asunto(s)

Simulación por Computador , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Helicobacter pylori/enzimología , Vectores Genéticos/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Helicobacter pylori/efectos de los fármacos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Recombinantes/aislamiento & purificación , Homología Estructural de Proteína

RESUMEN

Acquired Hemophagocytic Lymphohistiocytosis is a rare and deadly syndrome resulting from an overactive immune system, with uncontrolled activation of macrophages and lymphocytes, hypercytokinemia, and systemic inflammatory response. A 75-year-old male presented with typical anginal pain and was diagnosed with the acute coronary syndrome, which required a percutaneous transluminal coronary angioplasty. Instead of resolving the symptoms, the patient began to exhibit pyrexia and worsening altered sensorium with progressing renal failure, anemia, thrombocytopenia and respiratory failure. This constellation of symptoms caused the patient to require mechanical ventilation and hemodialysis. Upon laboratory analysis, hyperferritinemia provided an indication to the diagnosis of acquired hemophagocytic lymphohistiocytosis. After the initiation of dexamethasone, the patient made a significant recovery and was discharged from the hospital.

RESUMEN

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd) newborn screening is still a matter of debate due to its highly heterogeneous birth prevalence and clinical expression, as well as, the lack of enough knowledge on its natural history. Herein, we describe the early natural clinical course and the underlying GDPD genotypes in infants with G6PDd detected by newborn screening and later studied in a single follow-up center. G6PDd newborns were categorized into three groups: group 1: hospitalized with or without neonatal jaundice (NNJ); group 2: non-hospitalized with NNJ; and group 3: asymptomatic. Frequencies of homozygous UGT1A1*28 (rs34983651) genotypes among G6PDd patients with or without NNJ were also explored. RESULTS: A total of 81 newborns (80 males, one female) were included. Most individuals (46.9%) had NNJ without other symptoms, followed by asymptomatic (42.0%) and hospitalized (11.1%) patients, although the hospitalization of only 3 of these patients was related to G6PDd, including NNJ or acute hemolytic anemia (AHA). Nine different G6PDd genotypes were found; the G6PD A-202A/376G genotype was the most frequent (60.5%), followed by the G6PD A-376G/968C (22.2%) and the Union-Maewo (rs398123546, 7.4%) genotypes. These genotypes produce a wide range of clinical and biochemical phenotypes with significant overlapping residual enzymatic activity values among class I, II or III variants. Some G6PD A-202A/376G individuals had enzymatic values that were close to the cutoff value (5.3 U/g Hb, 4.6 and 4.8 U/g Hb in the groups with and without NNJ, respectively), while others showed extremely low enzymatic values (1.1 U/g Hb and 1.4 U/g Hb in the groups with and without NNJ, respectively). Homozygosity for UGT1A1*28 among G6PDd patients with (11.9%, N = 5/42) or without (10.3%, N = 4/39) NNJ did not shown significant statistical difference (p = 0.611). CONCLUSION: Wide variability in residual enzymatic activity was noted in G6PDd individuals with the same G6PD genotype. This feature, along with a documented heterogeneous mutational spectrum, makes it difficult to categorize G6PD variants according to current WHO classification and precludes the prediction of complications such as AHA, which can occur even with > 10% of residual enzymatic activity and/or be associated with the common and mild G6PD A-376G/968C and G6PD A-202A/376G haplotypes.

Asunto(s)

Deficiencia de Glucosafosfato Deshidrogenasa , Ictericia Neonatal , Femenino , Genotipo , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Deficiencia de Glucosafosfato Deshidrogenasa/epidemiología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Haplotipos , Humanos , Recién Nacido , Masculino , Tamizaje Neonatal

RESUMEN

Acquired Hemophagocytic Lymphohistiocytosis is a rare and deadly syndrome resulting from an overactive immune system, with uncontrolled activation of macrophages and lymphocytes, hypercytokinemia, and systemic inflammatory response. A 75-year-old male presented with typical anginal pain and was diagnosed with the acute coronary syndrome, which required a percutaneous transluminal coronary angioplasty. Instead of resolving the symptoms, the patient began to exhibit pyrexia and worsening altered sensorium with progressing renal failure, anemia, thrombocytopenia and respiratory failure. This constellation of symptoms caused the patient to require mechanical ventilation and hemodialysis. Upon laboratory analysis, hyperferritinemia provided an indication to the diagnosis of acquired hemophagocytic lymphohistiocytosis. After the initiation of dexamethasone, the patient made a significant recovery and was discharged from the hospital.

Asunto(s)

Humanos , Masculino , Anciano , Linfohistiocitosis Hemofagocítica/complicaciones , Hiperferritinemia/diagnóstico , Dexametasona/uso terapéutico , Síndrome Coronario Agudo/complicaciones , Deficiencia de Glucosafosfato Deshidrogenasa

Asunto(s)

Humanos , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Enfermedad del Almacenamiento de Glucógeno Tipo I/diagnóstico por imagen , Dislipidemias

RESUMEN

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ±â€¯7.9 µM) and the K0.5 values determined for G6P (57.9 ±â€¯2.5 and 104.5 ±â€¯9.3 µM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.

Asunto(s)

Glucosafosfato Deshidrogenasa/metabolismo , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina/química , Sitios de Unión , Dominio Catalítico , Glucosa-6-Fosfato/química , Glucosa-6-Fosfato/metabolismo , Glucosafosfato Deshidrogenasa/clasificación , Glucosafosfato Deshidrogenasa/genética , Cinética , Simulación de Dinámica Molecular , NAD/metabolismo , NADP/química , NADP/metabolismo , Filogenia , Unión Proteica , Desnaturalización Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

G6PD deficiency is associated with erythrocyte deficiency in the X-chromosome enzyme. It causes a hematologic syndrome called hemolytic anemia that connects G6PD deficiency with X-linked condition. In the Middle East, including Saudi Arabia, G6PD deficiency is the most dominant genetic blood disorders. It results in higher rates of mortality and morbidity due to its incurable long-lasting nature and prevalence of physical and psychological incapacities. In this study, an attempt was made to evaluate the prevalence of G6PD deficiency among the Saudi population in Riyadh city. A cross-sectional retrospective study was conducted at King Saud University Medical City in Riyadh, Saudi Arabia. The population of the study comprised randomly chosen males and females who visited the hospital from January 2017 to January 2018. Statistical analyses were performed using SPSS, and descriptive analysis was used to find the frequency of G6PD-deficient patients. Out of the 209 patients, 62.2% were males (n=130) and 37.8% were females (n=79). Twenty males and 6 females were found to have G6PD deficiency, with the male to female ratio being 1:3. Out of the total 130 male participants, 20 patients were found to be enzyme deficient and 6 patients of 79 female patients were found to be G6PD deficient. There were 38.4% (n=10) patients with G6PD level <4 units/gram hemoglobin, 26.9% (n=7) patients had G6PD levels of 4.1­7.0 units/gram hemoglobin, and 34.6% (n=9) patients had >7 units/gram hemoglobin. Among the G6PD patients, 23.07% patients were severely anemic, and 5 (19.2%) patients were reported to have high bilirubin. The present study revealed the G6PD prevalence to be 12.4% among the Saudi population; this value is significantly higher than that found in France, Spain, India, and Singapore. In the Saudi population, males are more vulnerable to G6PD-deficient than females. Hence, attention should be paid to G6PD-deficient patients while prescribing antimalarial medication. Such patients may be advised to avoid certain foods to minimize the risk of having hemolytic episodes.

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A deficiência de G6PD está associada à deficiência de eritrócitos na enzima do cromossomo X. Causa uma síndrome hematológica chamada anemia hemolítica que conecta a deficiência de G6PD à condição ligada ao X. No Oriente Médio, incluindo a Arábia Saudita, a deficiência de G6PD é o distúrbio genético do sangue mais dominante. Isso resulta em maiores taxas de mortalidade e morbidade devido à sua natureza incurável e duradoura e à prevalência de incapacidades físicas e psicológicas. Neste estudo, foi feita uma tentativa de avaliar a prevalência de deficiência de G6PD entre a população saudita na cidade de Riade. Um estudo retrospectivo transversal foi realizado na cidade médica da Universidade King Saud, em Riade, na Arábia Saudita. A população do estudo compreendeu homens e mulheres escolhidos aleatoriamente que visitaram o hospital entre janeiro de 2017 e janeiro de 2018. As análises estatísticas foram realizadas com o SPSS e a análise descritiva foi utilizada para determinar a frequência de pacientes com deficiência de G6PD. Dos 209 pacientes, 62,2% eram do sexo masculino (n = 130) e 37,8% eram do sexo feminino (n = 79). Verificou-se que 20 homens e 6 mulheres apresentavam deficiência de G6PD, sendo a proporção homem/mulher de 1:3. Do total de 130 participantes do sexo masculino, 20 pacientes apresentaram deficiência de enzima e 6 de 79 pacientes do sexo feminino apresentaram deficiência de G6PD. Havia 38,4% (n = 10) pacientes com nível de G6PD < 4 unidades/grama de hemoglobina, 26,9% (n = 7) pacientes tinham níveis de G6PD de 4,1-7,0 unidades/grama de hemoglobina e 34,6% (n = 9) pacientes tinham > 7 unidades/grama de hemoglobina. Entre os pacientes com G6PD, 23,07% eram severamente anêmicos e cinco (19,2%) pacientes relataram ter alta bilirrubina. O presente estudo revelou que a prevalência de G6PD é de 12,4% na população saudita; esse valor é significativamente maior que o encontrado na França, Espanha, Índia e Cingapura. Na população saudita, os homens são mais vulneráveis à deficiência de G6PD do que as mulheres. Portanto, deve-se prestar atenção aos pacientes com deficiência de G6PD durante a prescrição de medicamentos antimaláricos. Esses pacientes podem ser aconselhados a evitar certos alimentos para minimizar o risco de episódios hemolíticos.

Asunto(s)

Glucosafosfato Deshidrogenasa , Hemólisis , Anemia Hemolítica

RESUMEN

Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes participating in this pathway have not yet been characterized in detail. The objective of the present work was to clone, purify, and biochemically and physicochemically characterize glucose-6-phosphate dehydrogenase (G6PD) from GDI. The gene was cloned and expressed as a tagged protein in E. coli to be purified by affinity chromatography. The native state of the G6PD protein in the solution was found to be a tetramer with optimal activity at pH 8.8 and a temperature between 37 and 50 °C. The apparent Km values for G6P and nicotinamide adenine dinucleotide phosphate (NADP+) were 63 and 7.2 µM, respectively. Finally, from the amino acid sequence a three-dimensional (3D) model was obtained, which allowed the arrangement of the amino acids involved in the catalytic activity, which are conserved (RIDHYLGKE, GxGGDLT, and EKPxG) with those of other species, to be identified. This characterization of the enzyme could help to identify new environmental conditions for the knowledge of the plant-microorganism interactions and a better use of GDI in new technological applications.

Asunto(s)

Clonación Molecular , Gluconacetobacter/enzimología , Glucosafosfato Deshidrogenasa/metabolismo , Escherichia coli/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Concentración de Iones de Hidrógeno , Cinética , NADP/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Temperatura

RESUMEN

Surface based on polyelectrolytes functionalized with amino acids onto amino-terminated solid surfaces of silicon wafers was prepared, with the purpose of evaluate the chemical functionality of the polyelectrolyte films in adsorption and catalytic activity of an enzyme. In this work, the adsorption of the enzyme glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (LmG6PD) was studied as model. The polyelectrolytes were obtained from poly (maleic anhydride-alt-vinylpyrrolidone) [poly(MA-alt-VP)] and functionalized with amino acids of different hydropathy index: glutamine (Gln), tyrosine (Tyr) and methionine (Met). The polyelectrolytes were adsorbed onto the amino-terminated silicon wafer at pH 3.5 and 4.5 and at low and high ionic strength. At low ionic strength and pH 3.5, the largest quantity of adsorbed polyelectrolyte was on the films containing glutamine moiety as the most hydrophilic amino acid in the side chain of polymer chain (5.88 mg/m2), whereas at high ionic strength and pH 4.5, the lowest quantity was in films containing tyrosine moiety in the side chain (1.88 mg/m2). The films were characterized by ellipsometry, contact angle measurements and atomic force microscopy (AFM). The polyelectrolyte films showed a moderate degree of hydrophobicity, the methionine derivative being the most hydrophobic film. With the aim of evaluate the effect of the amino acid moieties on the ability of the surface to adsorb enzymes, we study the activity of the enzyme on these surfaces. We observed that the polarity of the side chain of the amino acid in the polyelectrolyte affected the quantity of LmG6PD adsorbed, as well as its specific activity, showing that films prepared from poly(MA-alt-VP) functionalized with Met provide the best enzymatic performance. The results obtained demonstrated that the surfaces prepared from polyelectrolytes functionalized with amino acids could be an attractive and simple platform for the immobilization of enzymes, which could be of interest for biocatalysis applications.

Asunto(s)

Aminoácidos/metabolismo , Enzimas Inmovilizadas/metabolismo , Polielectrolitos/metabolismo , Adsorción , Espectroscopía de Resonancia Magnética con Carbono-13 , Glucosafosfato Deshidrogenasa/metabolismo , Leuconostoc/enzimología , NAD/biosíntesis , Polielectrolitos/química , Espectroscopía Infrarroja por Transformada de Fourier , Humectabilidad

RESUMEN

ABSTRACT Objective Hyperthyroidism causes many injuries in its target organs and the consequences are reflected systemically. As systemic alterations in hyperthyroidism at earlier stages have received partial attention, this study aimed to investigate systemic redox and inflammatory status at an early stage of T4-induced hyperthyroidism. Materials and methods Male Wistar rats were assigned to control and hyperthyroid groups (n = 7/group). The hyperthyroid group received L-thyroxine (12 mg/L) in their drinking water for 14 days whereas control group received only the vehicle. Body weight was measured on the 1st and 14th day of the protocol. On the 14th day, animals were anaesthetized. Blood was then collected from the retro-orbital venous plexus and then the animals were euthanised. The blood was separated into plasma and erythrocytes. Plasma was used to measure ROS levels, sulfhydryl compounds, IL-10, TNF-α and LDH levels; erythrocytes were used for the analysis of thioredoxin reductase activity, glutaredoxin content, and pentose cycle enzymes (total G6PD, G6PD and 6PGD). Results Hyperthyroid animals presented body weight gain and final body weight reduction, which was associated with increased ROS levels and decreased sulfhydryl content in plasma. Thioredoxin reductase activity, glutaredoxin content, and pentose cycle enzymes levels in erythrocytes, as well as IL-10, TNF-α and LDH plasma levels were unaltered. Conclusion Taken together, our results suggest an impairment in corporal mass associated with systemic oxidative stress at this stage of hyperthyroidism. Meanwhile, the pentose cycle was not influenced and systemic inflammation and tissue damage seem to be absent at this stage of hyperthyroidism.

Asunto(s)

Animales , Masculino , Ratas , Estrés Oxidativo/efectos de los fármacos , Eritrocitos/metabolismo , Hipertiroidismo/metabolismo , Oxidación-Reducción , Pentosas , Tiroxina , Ratas Wistar , Modelos Animales de Enfermedad , Eritrocitos/efectos de los fármacos , Hipertiroidismo/sangre , Antioxidantes/metabolismo