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1.
Toxicol Lett ; 320: 64-72, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31794810

RESUMO

Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments.


Assuntos
Acetilcolinesterase/sangue , Acetilcolinesterase/metabolismo , Encéfalo/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Reativadores da Colinesterase/farmacocinética , Eritrócitos/efeitos dos fármacos , Imidazóis/farmacocinética , Oximas/farmacocinética , Compostos de Piridínio/farmacocinética , Células A549 , Animais , Encéfalo/enzimologia , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Reativadores da Colinesterase/administração & dosagem , Reativadores da Colinesterase/toxicidade , Relação Dose-Resposta a Droga , Eritrócitos/enzimologia , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/metabolismo , Células Hep G2 , Humanos , Imidazóis/administração & dosagem , Imidazóis/toxicidade , Injeções Intramusculares , Masculino , Dose Máxima Tolerável , Camundongos Endogâmicos ICR , Oximas/administração & dosagem , Oximas/toxicidade , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/toxicidade , Medição de Risco , Distribuição Tecidual
2.
Oxid Med Cell Longev ; 2019: 1983975, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827670

RESUMO

Objective: Cholesterol oxidation products have an established proatherogenic and cytotoxic effect. An increased exposure to these substances may be associated with the development of atherosclerosis and cancers. Relatively little, though, is known about the effect of phytosterol oxidation products, although phytosterols are present in commonly available and industrial food products. Thus, the aim of the research was to assess the effect of 5α,6α-epoxyphytosterols, which are important phytosterol oxidation products, on redox state in rats. Material and Methods: The animals were divided into 3 groups and exposed to nutritional sterols by receiving feed containing 5α,6α-epoxyphytosterols (ES group) and 5α,6α-epoxycholesterol (Ech group) or sterol-free feed (C group). The levels of malondialdehyde (MDA), conjugated dienes (CD), and ferric reducing antioxidant potential (FRAP) were assayed in the plasma; anti-7-ketocholesterol antibodies and activity of paraoxonase-1 (PON1) were determined in serum, whereas the activity of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), S-glutathione transferase (GST), and superoxide dismutase (SOD) were assayed in RBCs. Results: During the experiment, the levels of lipid peroxidation products increased, such as CD and anti-7-ketocholesterol antibodies. At the same time, the plasma levels of FRAP and serum activity of PON1 decreased alongside the reduced activity of GPx, GR, and SOD in RBCs. There was no effect of the studied compounds on the plasma MDA levels or on the activity of CAT and GST in RBCs. Conclusions: Both 5α,6α-epoxyphytosterols and 5α,6α-epoxycholesterols similarly dysregulate the redox state in experimental animal model and may significantly impact atherogenesis.


Assuntos
Colesterol/análogos & derivados , Dieta , Estresse Oxidativo/efeitos dos fármacos , Fitosteróis/farmacologia , Animais , Anticorpos/sangue , Antioxidantes/química , Arildialquilfosfatase/sangue , Catalase/metabolismo , Colesterol/farmacologia , Eritrócitos/citologia , Eritrócitos/enzimologia , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Cetocolesteróis/imunologia , Masculino , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo
3.
Mutat Res ; 846: 403072, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31585629

RESUMO

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59-) or mutant reticulocyte frequencies (RETCD59-) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.


Assuntos
Benzofenoneídio/toxicidade , Carcinógenos/toxicidade , Ensaio Cometa , Proteínas de Membrana/genética , Testes para Micronúcleos , Ativação Metabólica , Animais , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/ultraestrutura , Masculino , Microssomos Hepáticos/enzimologia , Testes de Mutagenicidade , Mutação , Estudo de Prova de Conceito , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacos , Reticulócitos/ultraestrutura
4.
Environ Toxicol Pharmacol ; 72: 103244, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31557707

RESUMO

The glucose metabolism in the pentose cycle is essential to the source of NADPH. Deficiency of these enzymes have been linked to depression and psychotic disorders. Depression is an increasingly prevalent mental disorder which may cause loss of labor. Antidepressant drugs are commonly employed in treatments of mood disorders and anxiety treatment. The purpose of this study is to investigate the effects of aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol on the activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD) enzymes purified from human erythrocytes. It was found that aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol show effective inhibitor properties on purified G6PD and 6PGD enzymes. The IC50 values of these drugs were found in the range of 26.34 µM-5.78 mM for 6PGD and 16.26 µM-3.85 mM for G6PD. The Ki values of the drugs were found in the range of 30.21 ± 4.31 µM-4.51 ± 1.83 mM for 6PGD and 14.12 ± 3.48 µM-4.98 ± 1.14 mM for G6PD. Usage of drugs with significant biological effects may be a hazard in some conditions.


Assuntos
Antidepressivos/farmacologia , Eritrócitos/efeitos dos fármacos , Glucosefosfato Desidrogenase/antagonistas & inibidores , Via de Pentose Fosfato/efeitos dos fármacos , Fosfogluconato Desidrogenase/antagonistas & inibidores , Aripiprazol/farmacologia , Citalopram/farmacologia , Eritrócitos/enzimologia , Haloperidol/farmacologia , Humanos , Mirtazapina/farmacologia
5.
Nutrients ; 11(8)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357662

RESUMO

Glutathione transferase P1-1 (GSTP1-1) is expressed in some human tissues and is abundant in mammalian erythrocytes (here termed e-GST). This enzyme is able to detoxify the cell from endogenous and exogenous toxic compounds by using glutathione (GSH) or by acting as a ligandin. This review collects studies that propose GSTP1-1 as a useful biomarker in different fields of application. The most relevant studies are focused on GSTP1-1 as a biosensor to detect blood toxicity in patients affected by kidney diseases. In fact, this detoxifying enzyme is over-expressed in erythrocytes when unusual amounts of toxins are present in the body. Here we review articles concerning the level of GST in chronic kidney disease patients, in maintenance hemodialysis patients and to assess dialysis adequacy. GST is also over-expressed in autoimmune disease like scleroderma, and in kidney transplant patients and it may be used to check the efficiency of transplanted kidneys. The involvement of GSTP in the oxidative stress and in other human pathologies like cancer, liver and neurodegenerative diseases, and psychiatric disorders is also reported. Promising applications of e-GST discussed in the present review are its use for monitoring human subjects living in polluted areas and mammals for veterinary purpose.


Assuntos
Exposição Ambiental/efeitos adversos , Monitoramento Ambiental/métodos , Poluentes Ambientais/efeitos adversos , Eritrócitos/efeitos dos fármacos , Glutationa S-Transferase pi/sangue , Animais , Biomarcadores/sangue , Eritrócitos/enzimologia , Glutationa S-Transferase pi/genética , Nível de Saúde , Indicadores Básicos de Saúde , Humanos , Estresse Oxidativo/efeitos dos fármacos , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco
6.
Chem Biol Interact ; 310: 108735, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276662

RESUMO

Organophosphates (OPs) irreversibly inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The reactivation of these inhibited enzymes is paramount for their normal function. Present study evaluates reactivation potency of two newly developed oximes, K456 and K733, against paraoxon (POX)-inhibited human-RBC-AChE and human-plasma-BChE in comparison to reported reactivator, pralidoxime (2-PAM). In vitro studies showed higher intrinsic toxicities of both oximes than 2-PAM for AChE. No substantial reactivation of hBChE was noted by tested concentration. Contrary to 2-PAM, the in silico study predicted lower binding free energies for both oximes. However, the detailed interaction study revealed inability of oximes to interact with catalytic anionic site of AChE and hBChE in contrast to 2-PAM. Both in vitro and in silico studies conclude that K456 and K733 are unlikely to be used as reactivators of paraoxon-inhibited AChE or BChE.


Assuntos
Inibidores da Colinesterase/farmacologia , Reativadores da Colinesterase/farmacologia , Oximas/farmacologia , Paraoxon/antagonistas & inibidores , Compostos de Piridínio/farmacologia , Acetilcolinesterase/química , Butirilcolinesterase/química , Eritrócitos/enzimologia , Humanos , Paraoxon/farmacologia
7.
Chem Biol Interact ; 311: 108746, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31301288

RESUMO

Utilizing food additives at their optimized concentration is believed to be relatively safe, but their combinatorial effects remain largely unexplored. The influence of mixed food additives on the macromolecules may be altered by synergistic or antagonistic effects. It is previously shown that curcumin enhances the catalase activity by affecting its structural pocket in the active site. The aim of this study was to investigate the combination effects of food colorants sunset yellow FCF (SNY) and curcumin on the activation and/or inactivation of catalase activity using multispectral (fluorescence, FTIR, and UV-vis) analysis and simultaneous docking simulations. Kinetic studies demonstrated that SNY could significantly decrease catalase activity through a non-competitive inhibition mechanism. Fluorescence data indicated that SNY reduces intrinsic emission of catalase via a static quenching mechanism. Thermodynamic and molecular docking investigations suggested that catalase has one binding site for SNY, and hydrogen binding plays a main role in the binding reaction of catalase -SNY complex. Molecular dynamic simulation data indicated that the curcumin binding to the cavity, in the middle of the catalase helical domain, facilitates SNY binding to the enzyme pocket. For this purpose, the equilibrium dialysis system was used to study the stability and reversibility of SNY-catalase in the absence or presence of curcumin. The obtained data indicated that the binding of SNY-catalase is reversible and the stability of the complex is time-dependent. However, curcumin could make the complex more stable enhancing the SNY inhibition of catalase activity.


Assuntos
Compostos Azo/química , Catalase/metabolismo , Curcumina/química , Corantes de Alimentos/química , Compostos Azo/metabolismo , Sítios de Ligação , Catalase/antagonistas & inibidores , Domínio Catalítico , Curcumina/metabolismo , Eritrócitos/enzimologia , Corantes de Alimentos/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Termodinâmica
8.
Methods Mol Biol ; 1988: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147928

RESUMO

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.


Assuntos
Bioensaio/métodos , Citosol/enzimologia , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Centrifugação com Gradiente de Concentração , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica , Eritrócitos/enzimologia , Corantes Fluorescentes/metabolismo , Humanos , Inibidores de Proteassoma/farmacologia , Especificidade por Substrato/efeitos dos fármacos
9.
Aquat Toxicol ; 212: 154-161, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31128416

RESUMO

Elevated concentrations of nitrite develop occasionally in various aquatic habitats and aquaculture facilities, providing a potential danger for freshwater fish that take up nitrite via the gill chloride uptake mechanism. We studied the uptake, effects and metabolism of nitrite in blood, heart and skeletal muscle at two temperatures in striped catfish Pangasianodon hypophthalmus, a facultative air-breathing fish that is heavily cultivated in Southeast Asia. Exposure to 0.8 mM ambient nitrite increased blood [nitrite] and [methaemoglobin] (metHb) to high values at day 1, but values subsequently decreased towards controls at day 7. Blood [nitrite] and metHb content were unexpectedly higher at 27 °C (∼1.2 mM; 69% at day 1) than at 33 °C (∼0.9 mM; 55%), reflecting a lower nitrite uptake at the highest temperature, possibly via an increased reliance on air-breathing relative to water-breathing with temperature increase. A large fraction of the nitrite taken up was effectively eliminated by being detoxified to nitrate. Further, erythrocyte metHb reductase activity was increased during nitrite exposure, efficiently reducing metHb to functional haemoglobin. The uptake of nitrite into white skeletal musculature (main part of the fish) was much lower than into heart tissue. While heart [nitrite] was close to blood plasma levels, muscle [nitrite] peaked at ∼0.2 mM at day 1 and subsequently declined to ∼0.05 mM at day 7, which is below levels reported in various commercial cured meat products. Nitrite was partly metabolized to iron-nitrosyl, S-nitroso and N-nitroso compounds. The increase in nitros(yl)ated compounds was marginal in skeletal muscle and more pronounced in heart tissue.


Assuntos
Peixes-Gato , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Nitritos/metabolismo , Nitritos/toxicidade , Temperatura , Aclimatação , Animais , Peixes-Gato/metabolismo , Cloretos/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Água Doce/química , Coração/efeitos dos fármacos , Hemoglobinas/metabolismo , Metemoglobina/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miocárdio/química , Nitritos/sangue , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidade
10.
Einstein (Sao Paulo) ; 17(2): eAO4328, 2019 May 02.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31066790

RESUMO

OBJECTIVE: To compare the enzyme activity of different presentations of papain solution to validate in-house preparations. METHODS: Two papain solutions were prepared, and the third presentation was a commercial solution. Tests were carried out with samples of red cells typed as weak RhD. RESULTS: In-house prepared papain solutions showed similar enzyme reactivity, and statistically no differences compared to the enzyme activity of the commercial solution. CONCLUSION: Evaluating the cost-benefit ratio, the in-house prepared papain solutions present more economic advantages, and can be incorporated into immunohematological routines as a way to cope with periods of financial crisis and cost-containment policies.


Assuntos
Eritrócitos/enzimologia , Testes Hematológicos/normas , Papaína/química , Peptídeo Hidrolases/química , Soluções/normas , Testes de Aglutinação/métodos , Testes Hematológicos/economia , Humanos , Papaína/economia , Peptídeo Hidrolases/economia , Reprodutibilidade dos Testes , Sistema do Grupo Sanguíneo Rh-Hr/química , Sistema do Grupo Sanguíneo Rh-Hr/economia , Soluções/economia , Fatores de Tempo
11.
mSphere ; 4(3)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068431

RESUMO

The human malaria parasite Plasmodium falciparum causes disease as it replicates within the host's erythrocytes. We have found that an erythrocyte serine hydrolase, acylpeptide hydrolase (APEH), accumulates within developing asexual parasites. Internalization of APEH was associated with a proteolytic event that reduced the size of the catalytic polypeptide from 80 to 55 kDa. A triazole urea APEH inhibitor, termed AA74-1, was employed to characterize the role of parasite-internalized APEH. In cell lysates, AA74-1 was a potent and highly selective inhibitor of both host erythrocyte and parasite-internalized APEH. When added to cultures of ring-stage parasites, AA74-1 was a poor inhibitor of replication over one asexual replication cycle; however, its potency increased dramatically after a second cycle. This enhancement of potency was not abrogated by the addition of exogenous isopentenyl pyrophosphate, the sole essential product of apicoplast metabolism. High-potency inhibition of parasite growth could be effected by adding AA74-1 to schizont-stage parasites, which resulted in parasite death at the early trophozoite stage of the ensuing replication cycle. Analysis of APEH inhibition in intact cultured cells revealed that host erythrocyte APEH, but not the parasite-internalized APEH pool, was inhibited by exogenous AA74-1. Our data support a model for the mode of parasiticidal activity of AA74-1 whereby sustained inactivation of host erythrocyte APEH is required prior to merozoite invasion and during parasite asexual development. Together, these findings provide evidence for an essential catalytic role for parasite-internalized APEH.IMPORTANCE Nearly half a million deaths were attributed to malaria in 2017. Protozoan parasites of the genus Plasmodium cause disease in humans while replicating asexually within the host's erythrocytes, with P. falciparum responsible for most of the mortality. Understanding how Plasmodium spp. have adapted to their unique host erythrocyte environment is important for developing malaria control strategies. Here, we demonstrate that P. falciparum coopts a host erythrocyte serine hydrolase termed acylpeptide hydrolase. By showing that the parasite requires acylpeptide hydrolase activity for replication, we expand our knowledge of host cell factors that contribute to robust parasite growth.


Assuntos
Eritrócitos/enzimologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/fisiologia , Reprodução Assexuada , Células Cultivadas , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo
12.
Biochem Pharmacol ; 166: 1-11, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071329

RESUMO

Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, thus maintaining ion cellular homeostasis. The present study aims to investigate the effect of several natural flavonoids on PMCA both in isolated protein systems and in living cells, and to establish the relationship between flavonoid structure and inhibitory activity on PMCA. Our results show that natural flavonoids inhibited purified and membranous PMCA with different effectiveness: quercetin and gossypin were the most potent and their inhibition mechanisms seem to be different, as quercetin does not prevent ATP binding whereas gossypin does. Moreover, PMCA activity was inhibited in human embryonic kidney cells which transiently overexpress PMCA, suggesting that the effects observed on isolated systems could occur in a complex structure like a living cell. In conclusion, this work reveals a novel molecular mechanism through which flavonoids inhibit PMCA, which leads to Ca2+ homeostasis and signaling alterations in the cell.


Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Células HEK293 , Humanos
13.
Toxicol Appl Pharmacol ; 371: 12-19, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928402

RESUMO

The increment of eryptosis in lead-exposed workers has been associated with oxidative stress, having as the main mediator [Ca2+]i. However, other molecules could participate as signals, such as PLA2 and SMase, which have been proposed to increase PGE2 and ceramides, both involved in the increment of PS externalization due to osmotic stress. To study the role of these enzymes in lead intoxication, we studied 30 lead exposed workers and 27 non-lead exposed individuals. We found, compared to non-exposed subjects, lead intoxication characterized by high blood lead concentration (median = 39.1 µg/dL), and low δ-ALAD activity (median = 348 nmol of porphobilinogen/h/mL); oxidative stress with high lipid peroxidation (median = 1.31 nmol of malondialdehyde/mL) and low TAC (median = 370 mM Trolox equivalents); a higher enzymatic activity of PLA2 (median = 518 AFU/mg) and SMase (median = 706 AFU/mg) and higher eryptosis (median = 0.92% PS externalization). Correlation and conditional probability analyses permit to associate oxidative stress and eryptosis with high PLA2 activity. However, high SMase activity was only associated with PLA2 activity. The role of these enzymes in the signal path to eryptosis induced by oxidative stress in lead-exposed workers is discussed.


Assuntos
Poluentes Ambientais/efeitos adversos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Intoxicação por Chumbo/etiologia , Chumbo/efeitos adversos , Exposição Ocupacional/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Fosfolipases A2/sangue , Esfingomielina Fosfodiesterase/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Poluentes Ambientais/sangue , Eritrócitos/enzimologia , Eritrócitos/patologia , Humanos , Chumbo/sangue , Intoxicação por Chumbo/sangue , Intoxicação por Chumbo/enzimologia , Intoxicação por Chumbo/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Pessoa de Meia-Idade , Sintase do Porfobilinogênio/sangue , Medição de Risco , Transdução de Sinais , Adulto Jovem
14.
Blood Adv ; 3(8): 1244-1254, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30987969

RESUMO

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is 1 potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wild-type siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal 2 novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.


Assuntos
Diferenciação Celular/genética , Eritrócitos/enzimologia , Eritropoese/genética , Hidroximetilglutaril-CoA Sintase , Mutação de Sentido Incorreto , Terpenos/metabolismo , Peixe-Zebra , Substituição de Aminoácidos , Animais , Colesterol/biossíntese , Colesterol/genética , Fator de Transcrição GATA1/biossíntese , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
15.
J Clin Pathol ; 72(6): 393-398, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30918013

RESUMO

Adenylate kinase (AK) deficiency is a rare erythroenzymopathy associated with hereditary nonspherocytic haemolytic anaemia along with mental/psychomotor retardation in few cases. Diagnosis of AK deficiency depends on the decreased level of enzyme activity in red cell and identification of a mutation in the AK1 gene. Until, only eight mutations causing AK deficiency have been reported in the literature. We are reporting two novel missense mutation (c.71A > G and c.413G > A) detected in the AK1 gene by next-generation sequencing (NGS) in a 6-year-old male child from India. Red cell AK enzyme activity was found to be 30% normal. We have screened a total of 32 family members of the patient and showed reduced red cell enzyme activity and confirm mutations by Sanger's sequencing. On the basis of Sanger sequencing, we suggest that the proband has inherited a mutation in AK1 gene exon 4 c.71A > G (p.Gln24Arg) from paternal family and exon 6 c.413G > A (p.Arg138His) from maternal family. Bioinformatics tools, such as SIFT, Polymorphism Phenotyping v.2, Mutation Taster, MutPred, also confirmed the deleterious effect of both the mutations. Molecular modelling suggests that the structural changes induced by p.Gln24Arg and p.Arg138His are pathogenic variants having a direct impact on the structural arrangement of the region close to the active site of the enzyme. In conclusion, NGS will be the best solution for diagnosis of very rare disorders leading to better management of the disease. This is the first report of the red cell AK deficiency from the Indian population.


Assuntos
Adenilato Quinase/genética , Anemia Hemolítica Congênita não Esferocítica/genética , Eritrócitos/enzimologia , Mutação de Sentido Incorreto , Adenilato Quinase/sangue , Adenilato Quinase/química , Adenilato Quinase/deficiência , Adulto , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Criança , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Hereditariedade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Índia , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade
16.
Artigo em Inglês | MEDLINE | ID: mdl-30901734

RESUMO

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 µm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ensaios Enzimáticos/métodos , Eritrócitos/enzimologia , Metiltransferases/sangue , Metiltransferases/metabolismo , Monitoramento de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Mercaptopurina/análogos & derivados , Mercaptopurina/metabolismo , Reprodutibilidade dos Testes , S-Adenosilmetionina/análise , S-Adenosilmetionina/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-30813224

RESUMO

The aim of this study was to verify whether eight-week-long swimming exercise training would evaluate the level of selected indicators of the pro-oxidant/antioxidant status in response to cold water in comparison with swimming under thermoneutral conditions in sedentary male and female elderly rats. The exercise-trained groups swam four min/day and five days a week during eight weeks of housing. Exercise was performed by swimming in glass tanks containing tap water maintained according to group at 5 °C and 36 °C. At the end of treatment (48 h after the last session), all rats were anaesthetized. The level of chosen biomarkers of oxidative stress and antioxidant enzyme activity was determined in the red blood cells and plasma. The results of study show that female rats seem to be better adapted to changing thermal conditions of the environment, developing not only morphological, but also antioxidant, defense mechanisms, mainly in the form of increased erythrocyte superoxide dismutase (SOD) activity and glutathione (GSH) concentration to restore the pro-oxidant/oxidant balance of the organism. Significantly higher concentrations of GSH were observed in the female rats of the group swimming in cold water (by 15.4% compared to the control group and by 20.5% in relation to the group of female rats swimming at 36 °C). In the group exposed to swimming training exercise in cold water, a significantly higher activity of SOD1 (by 13.4%) was found compared to the control group. On the other hand, the organs of ageing male rats show a reduced capacity to increase the metabolic response to low temperatures compared to female ones. In addition, it was demonstrated that cold exposure leads to an increase in lipid peroxidation in tissues. On the other hand, the repeated exposure to low levels of oxidative stress may result in some adaptive changes in organisms that help them to resist stress-induced damage.


Assuntos
Antioxidantes/metabolismo , Temperatura Baixa , Eritrócitos/metabolismo , Peroxidação de Lipídeos , Estresse Oxidativo , Natação/fisiologia , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Eritrócitos/enzimologia , Feminino , Temperatura Alta , Masculino , Condicionamento Físico Animal , Plasma/metabolismo , Ratos , Ratos Wistar
18.
Biochimie ; 160: 100-112, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30822441

RESUMO

Acetylcholinesterase (AChE) is the primary target of organophosphorus pesticides (OPs). Ellman's method using Acetylthiocholine (ATCh) is the standard approach for the detection of AChE activity. Though ATCh is a popular substrate, it has certain drawbacks as well. Because of these limitations, there is a need for the development of reliable and rapid assays for determination of AChE activity in cases of OP poisoning. In the present work, we have used 1-Naphthyl acetate (1-NA) as a fluorogenic substrate for the estimation of AChE activity of human erythrocytes. It is well known that due to inter-individual variation in AChE activity, the baseline value cannot be correctly predicted. Therefore, using 1-NA, we have developed a rapid, sensitive and baseline free assay for the estimation of AChE activity. The assay is based on reactivation and fluorescence quenching using a cocktail of oximes for the determination of cholinesterase activity in a post-exposure sample. Moreover, it is free from interference due to oximolysis which is an established limitation of ATCh. We feel that such an assay using 1-NA has the potential to be explored at the point of care for rapid detection of OP poisoning.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/envenenamento , Eritrócitos/enzimologia , Fluorescência , Naftóis/química , Intoxicação por Organofosfatos/diagnóstico , Praguicidas/toxicidade , Ensaios Enzimáticos , Proteínas Ligadas por GPI/metabolismo , Humanos , Intoxicação por Organofosfatos/enzimologia , Compostos Organofosforados/toxicidade
19.
J Biol Chem ; 294(17): 6809-6821, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850395

RESUMO

ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry was first detected in red blood cell (RBC) membranes, but the P4-ATPases responsible have not been directly determined. Using affinity-based MS, we show that ATP11C is the only abundant P4-ATPase phospholipid flippase in human RBCs, whereas ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. We also found that ATP11A and ATP11B are present at low levels. Mutations in the gene encoding ATP11C are responsible for blood and liver disorders, but the disease mechanisms are not known. Using heterologous expression, we show that the T415N substitution in the phosphorylation motif of ATP11C, responsible for congenital hemolytic anemia, reduces ATP11C expression, increases retention in the endoplasmic reticulum, and decreases ATPase activity by 61% relative to WT ATP11C. The I355K substitution in the transmembrane domain associated with cholestasis and anemia in mice was expressed at WT levels and trafficked to the plasma membrane but was devoid of activity. We conclude that the T415N variant causes significant protein misfolding, resulting in low protein expression, cellular mislocalization, and reduced functional activity. In contrast, the I355K variant folds and traffics normally but lacks key contacts required for activity. We propose that the loss in ATP11C phospholipid flippase activity coupled with phospholipid scramblase activity results in the exposure of phosphatidylserine on the surface of RBCs, decreasing RBC survival and resulting in anemia.


Assuntos
Adenosina Trifosfatases/metabolismo , Eritrócitos/enzimologia , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/genética , Animais , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Dobramento de Proteína
20.
Med Hypotheses ; 124: 40-41, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30798914

RESUMO

Modern diets have become increasingly rich in fructose, for example through the addition of high-fructose corn syrup to many foods and drinks. It has been suggested that this might lead to hepatotoxicity, including the development of non-alcoholic fatty liver disease. After entering hepatocytes via insulin-independent glucose transporter 2 transmembrane carrier proteins, fructose is phosphorylated to fructose-1-phosphate in a reaction catalysed by fructokinase (ketohexokinase). In turn, fructose-1-phosphate is hydrolysed by aldolase B to glyceraldehydes. Glyceraldehydes may enter gluconeogenesis via fructose-1,6-bisphosphate and fructose-6-phosphate; glyceraldehydes may also enter glycogenolysis via pyruvate. The last pathway involves conversion of pyruvate to acetyl-CoA. Alternatively, pyruvate may be converted, via the action of the hepatic lactate dehydrogenase isoenzyme LDH-5, into lactate. In liver damage, the LDH-5 isoenzyme becomes elevated, predominantly in serum/plasma. We therefore hypothesised that if dietary fructose is associated with hepatotoxicity, there should be a positive correlation between erythrocyte fructose-6-phosphate and plasma LDH-5. This hypothesis was tested by assaying venous blood samples taken from 39 patients at rest, three hours after eating. Quantitative Fourier transform infrared spectrometry following gel electrophoresis was used to assay erythrocyte fructose-6-phosphate levels. Similarly, plasma LDH-5 concentrations were spectrophotometrically analysed, using the pyruvate-lactate reaction, following electrophoretic separation of the LDH isoenzymes. A significant positive correlation was found between the two variables (r = 0.44, p = 0.0047). This result, which supports our hypothesis, is evidence in favour of the possibility that dietary fructose is associated with hepatotoxicity. In addition to being a marker of hepatic damage, LDH-5 may play a more direct epigenetic role in causing liver damage; acute hepatic injury is associated with nuclear translocation of LDH, causing the production of lactate from pyruvate in the nucleus; in turn, the lactate inhibits histone deacetylase and is associated with upregulation of genes associated with the damage response, leading to cell death.


Assuntos
Frutose/efeitos adversos , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Adulto , Animais , Epigênese Genética , Eritrócitos/enzimologia , Feminino , Frutoquinases/metabolismo , Gluconeogênese , Xarope de Milho Rico em Frutose/efeitos adversos , Humanos , Isoenzimas/metabolismo , Lactato Desidrogenase 5 , Fígado/enzimologia , Masculino , Camundongos , Fosforilação , Projetos Piloto , Espectroscopia de Infravermelho com Transformada de Fourier
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