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1.
Chem Pharm Bull (Tokyo) ; 69(9): 832-839, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34470947

RESUMO

Thiamine (vitamin B1), which is synthesized only in bacteria, fungi and plants and which humans should take with diet, participates in basic biochemical and physiological processes in a versatile way and its deficiency is associated with neurological problems accompanied by cognitive dysfunctions. The rat glioblastoma (C6) model was used, which was exposed to a limited environment and toxicity with glutamate. The cells were stressed by exposure to glutamate in the presence and absence of thiamine. The difference in cell proliferation was evaluated in the XTT assay. Oxidative stress (OS) markers malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels, as well as endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (GRP78), activating transcription factor-4 (ATF-4), and C/EBP homologous protein (CHOP) levels, were measured with commercial kits. Apoptosis determined by flow cytometry was confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining. At all concentrations, thiamine protects the cells and increased the viability against glutamate-induced toxicity. Thiamine also significantly decreased the levels of MDA, while increasing SOD and CAT levels. Moreover, thiamine reduced ER stress proteins' levels. Moreover, it lessened the apoptotic cell amount and enhanced the live-cell percentage in the flow cytometry and DAPI staining. As a result, thiamine may be beneficial nutritional support for individuals with a predisposition to neurodegenerative disorders due to its protective effect on glutamate cytotoxicity in glioblastoma cells by suppressing OS and ER stress.


Assuntos
Glioblastoma/tratamento farmacológico , Substâncias Protetoras/farmacologia , Tiamina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glioblastoma/induzido quimicamente , Glioblastoma/patologia , Ácido Glutâmico , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/química , Ratos , Tiamina/química , Células Tumorais Cultivadas
2.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502400

RESUMO

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.


Assuntos
Giardia lamblia/efeitos dos fármacos , Giardíase/tratamento farmacológico , Hidrolases/metabolismo , Animais , Antiprotozoários/farmacologia , Simulação por Computador , Cisteína/química , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Giardia lamblia/patogenicidade , Giardíase/imunologia , Tiomalato Sódico de Ouro/farmacologia , Humanos , Hidrolases/efeitos dos fármacos , Hidrolases/ultraestrutura , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol , Tiamina/análogos & derivados , Tiamina/farmacologia , Trofozoítos/efeitos dos fármacos
3.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360775

RESUMO

Coupling glycolysis and mitochondrial tricarboxylic acid cycle, pyruvate dehydrogenase (PDH) complex (PDHC) is highly responsive to cellular demands through multiple mechanisms, including PDH phosphorylation. PDHC also produces acetyl-CoA for protein acetylation involved in circadian regulation of metabolism. Thiamine (vitamin B1) diphosphate (ThDP) is known to activate PDH as both coenzyme and inhibitor of the PDH inactivating kinases. Molecular mechanisms integrating the function of thiamine-dependent PDHC into general redox metabolism, underlie physiological fitness of a cell or an organism. Here, we characterize the daytime- and thiamine-dependent changes in the rat brain PDHC function, expression and phosphorylation, assessing their impact on protein acetylation and metabolic regulation. Morning administration of thiamine significantly downregulates both the PDH phosphorylation at Ser293 and SIRT3 protein level, the effects not observed upon the evening administration. This action of thiamine nullifies the daytime-dependent changes in the brain PDHC activity and mitochondrial acetylation, inducing diurnal difference in the cytosolic acetylation and acetylation of total brain proteins. Screening the daytime dependence of central metabolic enzymes and proteins of thiol/disulfide metabolism reveals that thiamine also cancels daily changes in the malate dehydrogenase activity, opposite to those of the PDHC activity. Correlation analysis indicates that thiamine abrogates the strong positive correlation between the total acetylation of the brain proteins and PDHC function. Simultaneously, thiamine heightens interplay between the expression of PDHC components and total acetylation or SIRT2 protein level. These thiamine effects on the brain acetylation system change metabolic impact of acetylation. The changes are exemplified by the thiamine enhancement of the SIRT2 correlations with metabolic enzymes and proteins of thiol-disulfide metabolism. Thus, we show the daytime- and thiamine-dependent changes in the function and phosphorylation of brain PDHC, contributing to regulation of the brain acetylation system and redox metabolism. The daytime-dependent action of thiamine on PDHC and SIRT3 may be of therapeutic significance in correcting perturbed diurnal regulation.


Assuntos
Encéfalo/metabolismo , Cetona Oxirredutases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sirtuínas/metabolismo , Tiamina/farmacologia , Acetilação/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de Tempo
4.
Nutrients ; 13(7)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34371879

RESUMO

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.


Assuntos
Anti-Inflamatórios/farmacologia , Ácido Ascórbico/farmacologia , Hidrocortisona/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Sepse/tratamento farmacológico , Tiamina/farmacologia , Adulto , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Quimioterapia Combinada , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Sepse/metabolismo , Adulto Jovem
5.
Cent Nerv Syst Agents Med Chem ; 21(2): 125-129, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165417

RESUMO

BACKGROUND: Epilepsy, the second most frequent neurological disease, is a chronic disorder with a high lifetime prevalence. Therefore, various studies are needed to find new effective therapeutic agents to treat seizures or prevent their complications. In this study, we investigated the effects of thiamine, melatonin and their combination on pentylenetetrazol (PTZ)-induced tonic-clonic seizures in mice. METHODS: Male mice were randomly divided into six groups, including control, seizure control, diazepam, melatonin, thiamine and melatonin, and thiamine combination groups. Drugs were given orally in drinking water for 14 days. On the 15th day, the seizure was induced (except the control group) by intraperitoneal injection of PTZ. In all groups, the time between the injection and the start of the seizure (latency), and also the length of the seizure attack (duration), were measured in a 30-minute period. After measuring the latency and duration in all groups, mice were killed by CO2 Box and their brains were dissected to be analyzed for malondialdehyde (MDA) level as a marker of oxidative stress. RESULTS: The seizure duration was significantly lower in the groups of melatonin, thiamine and thiamine and melatonin combination compared to the seizure control group. The latency times in these groups were significantly greater than in the seizure control group. Moreover, MDA concentrations were lower in these groups compared to the seizure control group. CONCLUSION: Thiamine, melatonin and their combination can decrease the duration time of seizure and increase the latency period, which may result from inhibition of oxidative stress in the brain.


Assuntos
Melatonina , Animais , Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Masculino , Melatonina/uso terapêutico , Camundongos , Estresse Oxidativo , Pentilenotetrazol/uso terapêutico , Pentilenotetrazol/toxicidade , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Tiamina/farmacologia , Tiamina/uso terapêutico
6.
Biomed Pharmacother ; 141: 111823, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34147902

RESUMO

Here, we demonstrate that the two distinct formulations of our anti-sepsis drug candidate Rejuveinix (RJX), have a very favorable safety profile in Wistar Albino rats at dose levels comparable to the projected clinical dose levels. 14-day treatment with RJX-P (RJX PPP.18.1051) or RJX-B (RJX-B200702-CLN) similarly elevated the day 15 tissue levels of the antioxidant enzyme superoxide dismutase (SOD) as well as ascorbic acid in both the lungs and liver in a dose-dependent fashion. The activity of SOD and ascorbic acid levels were significantly higher in tissues of RJX-P or RJX-B treated rats than vehicle-treated control rats (p < 0.0001). There was no statistically significant difference between tissue SOD activity or ascorbic acid levels of rats treated with RJX-P vs. rats treated with RJX-B (p > 0.05). The observed elevations of the SOD and ascorbic acid levels were transient and were no longer detectable on day 28 following a 14-day recovery period. These results demonstrate that RJX-P and RJX-B are bioequivalent relative to their pharmacodynamic effects on tissue SOD and ascorbic acid levels. Furthermore, both formulations showed profound protective activity in a mouse model of sepsis. In agreement with the PD evaluations in rats and their proposed mechanism of action, both RJX-P and RJX-B exhibited near-identical potent and dose-dependent anti-oxidant and anti-inflammatory activity in the LPS-GalN model of ARDS and multi-organ failure in mice.


Assuntos
Ácido Ascórbico/química , Ácido Ascórbico/uso terapêutico , Sulfato de Magnésio/química , Sulfato de Magnésio/uso terapêutico , Niacinamida/química , Niacinamida/uso terapêutico , Ácido Pantotênico/química , Ácido Pantotênico/uso terapêutico , Piridoxina/química , Piridoxina/uso terapêutico , Riboflavina/química , Riboflavina/uso terapêutico , Sepse/tratamento farmacológico , Sepse/metabolismo , Tiamina/química , Tiamina/uso terapêutico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/química , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ácido Ascórbico/farmacologia , Cães , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Composição de Medicamentos , Feminino , Humanos , Lipopolissacarídeos/toxicidade , Sulfato de Magnésio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ácido Pantotênico/farmacologia , Piridoxina/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Riboflavina/farmacologia , Sepse/patologia , Superóxido Dismutase/metabolismo , Tiamina/farmacologia
7.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063830

RESUMO

Thiamine (vitamin B1) is essential for brain function because of the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. In order to compensate thiamine deficiency, several thiamine precursors with higher bioavailability were developed since the 1950s. Among these, the thioester benfotiamine (BFT) has been extensively studied and has beneficial effects both in rodent models of neurodegeneration and in human clinical studies. BFT has antioxidant and anti-inflammatory properties that seem to be mediated by a mechanism independent of the coenzyme function of ThDP. BFT has no adverse effects and improves cognitive outcome in patients with mild Alzheimer's disease (AD). Recent in vitro studies show that another thiamine thioester, dibenzoylthiamine (DBT) is even more efficient that BFT, especially with respect to its anti-inflammatory potency. Thiamine thioesters have pleiotropic properties linked to an increase in circulating thiamine concentrations and possibly in hitherto unidentified metabolites in particular open thiazole ring derivatives. The identification of the active neuroprotective derivatives and the clarification of their mechanism of action open extremely promising perspectives in the field of neurodegenerative, neurodevelopmental and psychiatric conditions.


Assuntos
Fármacos Neuroprotetores/farmacologia , Tiamina/análogos & derivados , Tiamina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Humanos , Neuroproteção/efeitos dos fármacos
8.
Chem Biol Interact ; 340: 109447, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771525

RESUMO

Accumulating evidences indicate that thiamine plays a vital role in the nervous system. However, questions exist as to how it causes epilepsy, neuronal damage, and antiepileptic mechanisms. The study looked at how the thiamine supplement impacted pentylenetetrazole (PTZ)-induced seizures in rats and pentylenetetrazole-induced neurotoxicity in the SH-SY5Y cell line. We used twenty-four male rats and they were randomly divided into 4 groups as control, saline (1 mL/kg/day serum physiologic) + PTZ, thiamine (50 mg/kg/day) + PTZ, and thiamine (50 mg/kg/day) for 10 days. PTZ (45 mg/kg) was given to activate the seizure on day 10. Memory efficiency was measured by using passive avoidance. The brain levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), caspase-3, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) were analyzed by using ELISA kits. SH-SY5Y cells were treated with/without thiamine for 1 h, followed by PTZ (30 µm) at a medium level to trigger neurotoxicity. Cell viability, total antioxidant status, total oxidant status, and apoptosis were assayed in the SH-SY5Y cells. Thiamine delayed the initiation of epileptic seizures and increased memory damage. In addition, 8-OHdG, caspase-3, NO, and cGMP levels were significantly reduced in the brain and prevented pentylenetetrazole-induced neurotoxicity, apoptosis, enhanced antioxidant, and reduced oxidant in SH-SY5Y cells. Thiamine dramatically altered seizures, memory loss, oxidative stress, and apoptosis. Thiamine has a preventative effect on PTZ-induced seizures in rats and PTZ-induced neurotoxicity in SH-SY5Y neuroblastoma cells. It could prevent oxidative stress and signaling of NO/cGMP. Thiamine supplement could be used as an additional therapeutic agent in epilepsy.


Assuntos
Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Convulsões/tratamento farmacológico , Tiamina/farmacologia , Animais , Anticonvulsivantes/farmacologia , Antioxidantes/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Masculino , Memória/efeitos dos fármacos , Neurônios/metabolismo , Pentilenotetrazol/farmacologia , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/metabolismo
9.
Expert Rev Anti Infect Ther ; 19(2): 129-135, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32809870

RESUMO

INTRODUCTION: COVID-19 disease progresses through a number of distinct phases. The management of each phase is unique and specific. The pulmonary phase of COVID-19 is characterized by an organizing pneumonia with profound immune dysregulation, activation of clotting, and a severe microvascular injury culminating in severe hypoxemia. The core treatment strategy to manage the pulmonary phase includes the combination of methylprednisolone, ascorbic acid, thiamine, and heparin (MATH+ protocol). The rationale for the MATH+ protocol is reviewed in this paper. AREAS COVERED: We provide an overview on the pathophysiological changes occurring in patients with COVID-19 respiratory failure and a treatment strategy to reverse these changes thereby preventing progressive lung injury and death. EXPERT OPINION: While there is no single 'Silver Bullet' to cure COVID-19, we believe that the severely disturbed pathological processes leading to respiratory failure in patients with COVID-19 organizing pneumonia will respond to the combination of Methylprednisone, Ascorbic acid, Thiamine, and full anticoagulation with Heparin (MATH+ protocol).We believe that it is no longer ethically acceptable to limit management to 'supportive care' alone, in the face of effective, safe, and inexpensive medications that can effectively treat this disease and thereby reduce the risk of complications and death.


Assuntos
Ácido Ascórbico/farmacologia , COVID-19 , Protocolos Clínicos , Heparina/farmacologia , Metilprednisolona/farmacologia , Tiamina/farmacologia , Anti-Inflamatórios/farmacologia , Anticoagulantes/farmacologia , COVID-19/sangue , COVID-19/tratamento farmacológico , COVID-19/metabolismo , COVID-19/fisiopatologia , Humanos , Gravidade do Paciente , SARS-CoV-2 , Vitaminas/farmacologia
10.
Chem Biol Interact ; 333: 109312, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33166511

RESUMO

Chlorpyrifos is a extensively used organophosphate pesticide (OP). In this study, we closely looked into neurotoxicity of CPF and effect of vitamin B1, by checking the levels of cholinesterases, determining the activity of parameters of oxidative stress, inflammation and also level of apoptotic regulator. The study was performed on a total of 80 male Japanese quails (Coturnix japonica), (two control and 6 experimental groups, n = 10). Three group of quails were given by gavage chlorpyrifos (CPF) for 7 consecutive days at doses of 1.50 mg/kg b.w., 3.00 mg/kg b.w., and 6.00 mg/kg b.w. Another three groups were treated with 10 mg/kg b.w. of vitamin B1 i.m. 30 min after CPF application (in above mentioned doses). Our study have proved that all doses of CPF significantly inhibited cholinesterases in brain, while vitamin B1 reactivated them. CPF has led to an increase in the concentration of malondialdehyde (MDA), and activity of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), while tiamin changed the activity of antioxidant enzymes: CAT, SOD, GST. CPF stimulated apoptosis by decreasing B-cell lymphoma (Bcl-2) in brain, while application of vitamin B1 caused an increase of this parameter. CPF amplified inflammatory effect by elevating levels of inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX-2). Thiamine proved its anti-inflammatory property by decreasing the expression of iNOS and interleukin-1(IL-1) and interleukin-6(IL-6). This study is highly pertinent because there is little defense currently available to humans and animals to prevent toxic effects of pesticides.


Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/enzimologia , Clorpirifos/toxicidade , Colinesterases/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Tiamina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Coturnix , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Masculino , Malondialdeído/metabolismo , Tiamina/administração & dosagem
11.
J Enzyme Inhib Med Chem ; 36(1): 122-129, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33187452

RESUMO

Oxythiamine (OT) and 3-deazathiamine (DAT) are the antimetabolites of thiamine. The aim of study was to compare the effects of OT and DAT pyrophosphates (-PP) on the kinetics of mammalian pyruvate dehydrogenase complex (PDHC) and the in vitro culture of HeLa cells. The kinetic study showed that 3-deazathiamine pyrophosphate (DATPP) was a much stronger competitive inhibitor (Ki = 0.0026 µM) of PDHC than OTPP (Ki = 0.025 µM). Both Ki values were much lower versus K m for thiamine pyrophosphate (0.06 µM). However, DATPP added to the culture medium for the HeLa cells culture did not hamper the rate of cell growth and showed not significant impact on the viability of the cells, whereas OTPP and OT showed a significant cytostatic effect. The differences between the thiamine antivitamins in their effect on cell growth in vitro may be due to differences in physicochemical properties and difficulty in DAT transport across the cell membrane.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Tiamina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Estrutura Molecular , Complexo Piruvato Desidrogenase/metabolismo , Relação Estrutura-Atividade , Tiamina/análogos & derivados , Tiamina/química , Células Tumorais Cultivadas
12.
ACS Synth Biol ; 9(12): 3202-3209, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33180466

RESUMO

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.


Assuntos
Sistemas CRISPR-Cas/genética , Pichia/genética , RNA Guia/metabolismo , Genes Reporter , Plasmídeos/genética , Plasmídeos/metabolismo , Riboflavina/biossíntese , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
13.
J Med Chem ; 63(22): 13745-13761, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33186038

RESUMO

The antiviral activity of nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside triphosphate analogues are presented, in which the γ-phosphate was covalently modified by a non-bioreversible, lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension assays using human immunodeficiency virus reverse transcriptase (HIV-RT) and three DNA-polymerases showed a high selectivity of these γ-modified nucleoside triphosphates to act as substrates for HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, ß, and γ. In contrast to d4TTP, the γ-modified d4TTPs showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these γ-ketobenzyl nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable γ-modified nucleoside triphosphate to increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of γ-ketobenzyl-d4TTPs was proven in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more importantly in thymidine kinase-deficient CD4+ T-cells.


Assuntos
Fármacos Anti-HIV/química , Pró-Fármacos/química , Fármacos Anti-HIV/farmacologia , Antivirais/química , Antivirais/farmacologia , Células Cultivadas , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Humanos , Polifosfatos/química , Polifosfatos/farmacologia , Pró-Fármacos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Linfócitos T/virologia , Tiamina/química , Tiamina/farmacologia
14.
Med Sci Monit ; 26: e924932, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33186340

RESUMO

BACKGROUND Studies have shown that thiamine intake is associated with cervical cancer, but the relationship between thiamine and HPV infection remains unclear. In the present study, we used the National Health and Nutrition Examination Survey (NHANES) database to investigate whether HPV infection was associated with thiamine intake. MATERIAL AND METHODS A total of 13 471 women ages 18-59 years were selected from the NHANES database from 2003 to 2016. Using thiamine intake as the independent variable, HPV infection as the dependent variable, and sociodemographic data and other data as the covariates, we analyzed the relationship between thiamine and HPV infection by conducting a weighted logistic regression model in a cross-sectional research design. RESULTS The two-piecewise linear model indicated the inflection point of thiamine intake was 2.07 mg. On the left side of the inflection point, the difference in the thiamine intake of log2 conversion was related to the difference of 0.82 in HPV infection, which means that the increase of every 1 unit increase in thiamine intake is associated with the decrease of the HPV infection by 18%. On the right side of the inflection point, we did not observe a correlation between HPV infection and thiamine intake. CONCLUSIONS Thiamine intake is negatively correlated with HPV infection. Intake of an appropriate amount of thiamine can prevent HPV infection. The best preventive effect can be achieved when the intake is about 2 mg, and excessive intake will not increase the preventive effect.


Assuntos
Análise de Dados , Inquéritos Nutricionais , Papillomaviridae/fisiologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Tiamina/administração & dosagem , Adulto , Feminino , Humanos , Modelos Lineares , Dinâmica não Linear , Tiamina/farmacologia , Estados Unidos/epidemiologia
15.
Exp Gerontol ; 141: 111097, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32987117

RESUMO

It is well known that patients with Alzheimer's disease (AD) have imbalances in blood thiamine concentrations and lower activity of thiamine-dependent enzymes. Benfotiamine, a more bioavailable thiamine analog, has been proposed as an alternative to counteract these changes related to thiamine metabolism. Thus, our study aimed to analyze the effects of benfotiamine supplementation on brain thiamine absorption, as well as on parameters related to neuronal energy metabolism and disease progression in an experimental model of sporadic AD induced by intracerebroventricular injection of streptozotocin (STZ) in rats. The supplementation with 150 mg/kg of benfotiamine for 30 days increased the concentrations of thiamine diphosphate in the hippocampus and entorhinal cortex. This led to an improvement in mitochondria enzymes and insulin signaling pathway, with inactivation of GSK3α/ß and ERK1/2, which are two tau-kinases related to the progression of AD, which could decrease tau hyperphosphorylation and apoptosis signaling. Besides, we observed an increased amount of Glun2b subunit of NMDA receptors, decreased inflammation, and improvement of cognitive deficit. Together, these results suggest that benfotiamine could be a potential therapeutic approach in the treatment of sporadic AD.


Assuntos
Doença de Alzheimer , Tiamina Pirofosfato , Doença de Alzheimer/tratamento farmacológico , Animais , Encéfalo , Cognição , Humanos , Ratos , Tiamina/análogos & derivados , Tiamina/farmacologia
16.
Nat Chem Biol ; 16(11): 1237-1245, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32839604

RESUMO

The natural antivitamin 2'-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2'-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.


Assuntos
Antibacterianos/metabolismo , Inibidores Enzimáticos/metabolismo , Tiamina/metabolismo , Transcetolase/antagonistas & inibidores , Sequência de Aminoácidos , Antibacterianos/farmacologia , Domínio Catalítico , Coenzimas/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Ácido Glutâmico/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Tiamina/farmacologia , Tiamina Pirofosfato/metabolismo , Transcetolase/genética
17.
RNA ; 26(11): 1743-1752, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32788323

RESUMO

The fission yeast Schizosaccharomyces pombe is an excellent model organism for the study of eukaryotic cellular physiology. The organism is genetically tractable and several tools to study the functions of individual genes are available. One such tool is regulatable gene expression and overproduction of proteins. Limitations of currently available overexpression systems include delay in expression after induction, narrow dynamic range, and system-wide changes due to induction conditions. Here I describe a new long noncoding RNA (lncRNA)-regulated, thiamine-inducible expression system that integrates lncRNA-based transcriptional interference at the fission yeast tgp1 promoter with the fast repression kinetics of the thiamine-repressible nmt1 promoter. This hybrid system has rapid induction kinetics, broad dynamic range, and tunable expression via thiamine concentration. The lncRNA-regulated thiamine-inducible system will be advantageous for the study of individual genes and for potential applications in the production of heterologous proteins in fission yeast.


Assuntos
RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Tiamina/farmacologia , Regulação Fúngica da Expressão Gênica , Vetores Genéticos , Cinética , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas , RNA Fúngico/genética
18.
Acta Diabetol ; 57(12): 1423-1433, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32656709

RESUMO

AIMS: Although diabetic retinopathy has long been considered a microvascular complication, retinal neurodegeneration and inflammation may precede its clinical manifestations. Despite all research efforts, the primary treatment options remain laser photocoagulation and anti-vascular endothelial growth factor (VEGF) intravitreal injections, both aggressive and targeting the late stages of the disease. Medical treatments addressing the early phases of diabetic retinopathy are therefore needed. We aimed at verifying if thiamine and fenofibrate protect the cells of the inner blood-retinal barrier from the metabolic stress induced by diabetic-like conditions. METHODS: Human microvascular endothelial cells (HMECs), retinal pericytes (HRPs) and Müller cells (MIO-M1) were cultured in intermittent high glucose (intHG) and/or hypoxia, with addition of fenofibrate or thiamine. Modulation of adhesion molecules and angiogenic factors was addressed. RESULTS: Integrins ß1/αVß3 and ICAM1 were upregulated in HMECs/HRPs cultured in diabetic-like conditions, as well as metalloproteases MMP2/9 in HRP, with a reduction in their inhibitor TIMP1; MMP2 increased also in HMEC, and TIMP1 decreased in MIO-M1. VEGF and HIF-1α were strongly increased in HMEC in intHG + hypoxia, and VEGF also in HRP. Ang-1/2 augmented in HMEC/MIO-M1, and MCP-1 in HRP/MIO-M1 in intHG + hypoxia. Thiamine was able to normalize all such abnormal modulations, while fenofibrate had effects in few cases only. CONCLUSIONS: We suggest that endothelial cells and pericytes are more affected than Müller cells by diabetic-like conditions. Fenofibrate shows a controversial behavior, potentially positive on Müller cells and pericytes, but possibly detrimental to endothelium, while thiamine confirms once more to be an effective agent in reducing diabetes-induced retinal damage.


Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fenofibrato/farmacologia , Glucose/farmacologia , Hipóxia/patologia , Tiamina/farmacologia , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Retinopatia Diabética/patologia , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Hipóxia/complicações , Hipóxia/metabolismo , Modelos Biológicos , Pericitos/efeitos dos fármacos , Pericitos/patologia , Retina/efeitos dos fármacos , Retina/patologia
20.
Nutrients ; 12(6)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517031

RESUMO

Diabetes mellitus-related morbidity and mortality is a rapidly growing healthcare problem, globally. Several nutraceuticals exhibit potency to target the pathogenesis of diabetes mellitus. The antidiabetic effects of compounds of garlic have been extensively studied, however, limited data are available on the biological effects of a certain garlic component, allithiamine. In this study, allithiamine was tested using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The non-cytotoxic concentration of allithiamine was determined based on MTT, apoptosis, and necrosis assays. Subsequently, cells were divided into three groups: incubating with M199 medium as the control; or with 30 mMol/L glucose; or with 30 mMol/L glucose plus allithiamine. The effect of allithiamine on the levels of advanced glycation end-products (AGEs), activation of NF-κB, release of pro-inflammatory cytokines including IL-6, IL-8, and TNF-α, and H2O2-induced oxidative stress was investigated. We found that in the hyperglycaemia-induced increase in the level of AGEs, pro-inflammatory changes were significantly suppressed by allithiamine. However, allithiamine could not enhance the activity of transketolase, but it exerts a potent antioxidant effect. Collectively, our data suggest that allithiamine could alleviate the hyperglycaemia-induced endothelial dysfunction due to its potent antioxidant and anti-inflammatory effect by a mechanism unrelated to the transketolase activity.


Assuntos
Anti-Inflamatórios , Antioxidantes , Endotélio Vascular/fisiopatologia , Alho/química , Hiperglicemia/tratamento farmacológico , Hiperglicemia/fisiopatologia , Fitoterapia , Tiamina/análogos & derivados , Citocinas/metabolismo , Endotélio Vascular/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tiamina/isolamento & purificação , Tiamina/farmacologia , Tiamina/uso terapêutico , Transcetolase/metabolismo
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