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1.
J Agric Food Chem ; 69(44): 13246-13254, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34702032

RESUMO

There is limited information available about the physiological content of glyceraldehyde, a precursor of toxic advanced glycation end products. The conventional derivatization method for aldoses using 1-phenyl-3-methyl-5-pyrazolone did not allow reproducible quantification of glyceraldehyde due to the instability of glyceraldehyde compared to other aldoses. We optimized the derivatization condition to achieve high and reproducible recovery of derivatives for liquid chromatography tandem mass spectrometry quantification. Based on the stability of glyceraldehyde during sample preparation and high recovery of spiked standard, the present method provides reproducible quantification of glyceraldehyde in the body. The glyceraldehyde contents in fasting conditions in the rodent liver (mice: 50.0 ± 3.9 nmol/g; rats: 35.5 ± 4.9 nmol/g) were higher than those in plasma (9.4 ± 1.7 and 7.2 ± 1.2 nmol/mL). The liver glyceraldehyde levels significantly increased after food consumption (p < 0.05) but remained constant in the plasma. High fat diet feeding significantly increased plasma glyceraldehyde levels in mice (p < 0.005). In healthy human volunteers, the plasma glyceraldehyde levels remained unchanged after the consumption of steamed rice. In patients with type 2 diabetes, the plasma glyceraldehyde level was positively correlated with the plasma glucose level (r = 0.84; p < 0.0001).


Assuntos
Diabetes Mellitus Tipo 2 , Gliceraldeído , Animais , Jejum , Produtos Finais de Glicação Avançada , Humanos , Camundongos , Ratos , Roedores
2.
Cells ; 10(8)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34440878

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive regression and memory loss. Dysfunctions of both glucose metabolism and mitochondrial dynamics have been recognized as the main upstream events of the degenerative processes leading to AD. It has been recently found that correcting cell metabolism by providing alternative substrates can prevent neuronal injury by retaining mitochondrial function and reducing AD marker levels. Here, we induced an AD-like phenotype by using the glycolysis inhibitor glyceraldehyde (GA) and explored whether L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid, LC) could mitigate neuronal damage, both in SH-SY5Y neuroblastoma cells and in rat primary cortical neurons. We have already reported that GA significantly modified AD marker levels; here we demonstrated that GA dramatically compromised cellular bioenergetic status, as revealed by glycolysis and oxygen consumption rate (OCR) evaluation. We found that LC ameliorated cell survival, improved OCR and ATP synthesis, prevented the loss of the mitochondrial membrane potential (Δψm) and reduced the formation of reactive oxygen species (ROS). Of note, the beneficial effect of LC did not rely on the glycolytic pathway rescue. Finally, we noticed that LC significantly reduced the increase in pTau levels induced by GA. Overall, these findings suggest that the use of LC can promote cell survival in the setting of the metabolic impairments commonly observed in AD. Our data suggest that LC may act by maintaining mitochondrial function and by reducing the pTau level.


Assuntos
Doença de Alzheimer/metabolismo , Carnitina/farmacologia , Gliceraldeído/toxicidade , Fármacos Neuroprotetores/farmacologia , Trifosfato de Adenosina/biossíntese , Doença de Alzheimer/induzido quimicamente , Animais , Sobrevivência Celular/efeitos dos fármacos , Glicólise , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas tau/metabolismo
3.
Nutrients ; 13(6)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072532

RESUMO

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.


Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Inflamação/prevenção & controle , Lactobacillus reuteri/metabolismo , Probióticos/farmacologia , Animais , Modelos Animais de Doenças , Gliceraldeído/análogos & derivados , Gliceraldeído/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Propano/metabolismo
4.
J Med Chem ; 64(11): 7483-7506, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34024109

RESUMO

Based on a multitarget strategy, a series of novel tacrine-pyrimidone hybrids were identified for the potential treatment of Alzheimer's disease (AD). Biological evaluation results demonstrated that these hybrids exhibited significant inhibitory activities toward acetylcholinesterase (AChE) and glycogen synthase kinase 3 (GSK-3). The optimal compound 27g possessed excellent dual AChE/GSK-3 inhibition both in terms of potency and equilibrium (AChE: IC50 = 51.1 nM; GSK-3ß: IC50 = 89.3 nM) and displayed significant amelioration on cognitive deficits in scopolamine-induced amnesia mice and efficient reduction against phosphorylation of tau protein on Ser-199 and Ser-396 sites in glyceraldehyde (GA)-stimulated differentiated SH-SY5Y cells. Furthermore, compound 27g exhibited eligible pharmacokinetic properties, good kinase selectivity, and moderate neuroprotection against GA-induced reduction in cell viability and neurite damage in SH-SY5Y-derived neurons. The multifunctional profiles of compound 27g suggest that it deserves further investigation as a promising lead for the prospective treatment of AD.


Assuntos
Inibidores da Colinesterase/química , Desenho de Fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Pirimidinonas/química , Tacrina/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Gliceraldeído/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Relação Estrutura-Atividade , Proteínas tau/metabolismo
5.
Cells ; 10(5)2021 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923186

RESUMO

Glyceraldehyde-derived advanced glycation end products (AGEs) play an important role in the pathogenesis of many diseases including cancer. Accumulation of intracellular AGEs could stimulate cancer induction and facilitate cancer progression. We evaluated the toxic effect of glyceraldehyde-derived intracellular AGEs on normal and malignant pancreatic ductal cells by assessing the cell viability, toxicity, and oxidative stress, followed by proteomic analysis. Our functional studies showed that pancreatic cancer cells (PANC-1 and MIA PaCa-2) were more resistant to glyceraldehyde treatment compared to normal pancreatic ductal epithelial cells (HPDE), while cytotoxicity effects were observed in all cell types. Furthermore, using 13C isotopic labeled glyceraldehyde, the proteomic data revealed a dose-dependent increment of the number of glycation adducts in both these cell types. HPDE cells showed a higher number of intracellular AGEs compared to cancer cells. At a molecular level, the glycations in the lysine residues of proteins showed a concurrent increase with the concentration of the glyceraldehyde treatment, while the arginine glycations appeared to be less affected by the glyceraldehyde doses. Further pathway analysis of these glycated proteins suggested that the glycated proteins participate in important biological processes that are major hallmarks of cancer initiation and progression, including metabolic processes, immune response, oxidative stress, apoptosis, and S100 protein binding.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/patologia , Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído/farmacologia , Estresse Oxidativo , Neoplasias Pancreáticas/patologia , Proteoma/metabolismo , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Sobrevivência Celular , Glicosilação , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteoma/análise , Células Tumorais Cultivadas
6.
Food Microbiol ; 98: 103720, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875197

RESUMO

Strains of Limosilactobacillus reuteri are used as starter and bioprotective cultures and contribute to the preservation of food through the production of fermentation metabolites lactic and acetic acid, and of the antimicrobial reuterin. Reuterin consists of acrolein and 3-hydroxypropionaldehyde (3-HPA), which can be further metabolized to 1,3-propanediol and 3-hydroxypropionic acid (3-HP). While reuterin has been the focus of many investigations, the contribution of 3-HP to the antimicrobial activity of food related reuterin-producers is unknown. We show that the antibacterial activity of 3-HP was stronger at pH 4.8 compared to pH 5.5 and 6.6. Gram-positive bacteria were in general more resistant against 3-HP and propionic acid than Gram-negative indicator strains including common food pathogens, while spoilage yeast and molds were not inhibited by ≤ 640 mM 3-HP. The presence of acrolein decreased the minimal inhibitory activity of 3-HP against E. coli indicating synergistic antibacterial activity. 3-HP was formed during the growth of the reuterin-producers, and by resting cells of L. reuteri DSM 20016. Taken together, this study shows that food-related reuterin producers strains synthesize a second antibacterial compound, which might be of relevance when strains are added as starter or bioprotective cultures to food products.


Assuntos
Anti-Infecciosos/farmacologia , Glicerol/metabolismo , Ácido Láctico/análogos & derivados , Lactobacillaceae/química , Ácido Acético/metabolismo , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Estabilidade de Medicamentos , Fermentação , Microbiologia de Alimentos , Gliceraldeído/análogos & derivados , Gliceraldeído/química , Gliceraldeído/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/química , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Lactobacillaceae/crescimento & desenvolvimento , Lactobacillaceae/metabolismo , Propano/química , Propano/metabolismo
7.
Sci Rep ; 11(1): 2959, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536515

RESUMO

Advanced glycation end-products (AGEs) are formed by the non-enzymatic reaction of sugars and proteins. Among the AGEs, glyceraldehyde-derived toxic AGEs (TAGE) are associated with various diseases, including diabetic complications such as diabetic retinopathy (DR). The risk of developing DR is strongly associated with poor glycemic control, which causes AGE accumulation and increases AGE-induced vascular permeability. We previously reported that Ras guanyl nucleotide releasing protein 2 (RasGRP2), which activates small G proteins, may play an essential role in the cell response to toxicity when exposed to various factors. However, it is not known whether RasGRP2 prevents the adverse effects of TAGE in vascular endothelial cells. This study observed that TAGE enhanced vascular permeability by disrupting adherens junctions and tight junctions via complex signaling, such as ROS and non-ROS pathways. In particular, RasGRP2 protected adherens junction disruption, thereby suppressing vascular hyper-permeability. These results indicate that RasGRP2 is an essential protective factor of vascular permeability and may help develop novel therapeutic strategies for AGE-induced DR.


Assuntos
Permeabilidade Capilar , Endotélio Vascular/patologia , Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Junções Aderentes/patologia , Retinopatia Diabética/patologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos
8.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140544, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971286

RESUMO

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) - described for other PLP-dependent enzymes when incubated with small aldehydes - or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction.


Assuntos
Trifosfato de Adenosina/química , Inibidores Enzimáticos/química , Gliceraldeído 3-Fosfato/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Fosfato de Piridoxal/química , Racemases e Epimerases/química , 2,3-Difosfoglicerato/química , 2,3-Difosfoglicerato/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Domínio Catalítico , Clonagem Molecular , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gliceraldeído/química , Gliceraldeído/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Fosfato de Piridoxal/metabolismo , Racemases e Epimerases/antagonistas & inibidores , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
9.
Nutrients ; 12(10)2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32992566

RESUMO

Nutritional factors can affect the risk of developing neurological disorders and their rate of progression. In particular, abnormalities of carbohydrate metabolism in diabetes mellitus patients lead to an increased risk of neurological disorders such as Alzheimer's disease (AD). In this study, we investigated the relationship between nervous system disorder and the pathogenesis of AD by exposing SH-SY5Y neuroblastoma cells to glyceraldehyde (GA). We previously reported that GA-derived toxic advanced glycation end products (toxic AGEs, TAGE) induce AD-like alterations including intracellular tau phosphorylation. However, the role of TAGE and their target molecules in the pathogenesis of AD remains unclear. In this study, we investigated the target protein for TAGE by performing two-dimensional immunoblot analysis with anti-TAGE antibody and mass spectrometry and identified ß-tubulin as one of the targets. GA treatment induced TAGE-ß-tubulin formation and abnormal aggregation of ß-tubulin, and inhibited neurite outgrowth in SH-SY5Y cells. On the other hand, glucose-derived AGEs were also involved in developing AD. However, glucose did not make abnormal aggregation of ß-tubulin and did not inhibit neurite outgrowth. Understanding the underlying mechanism of TAGE-ß-tubulin formation by GA and its role in neurodegeneration may aid in the development of novel therapeutics and neuroprotection strategies.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído/farmacologia , Neuroblastoma/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacologia , Doença de Alzheimer/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus , Progressão da Doença , Glucose , Humanos , Tubulina (Proteína)/genética
10.
Gut Microbes ; 12(1): 1788898, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32804011

RESUMO

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.


Assuntos
Clostridioides difficile/efeitos dos fármacos , Gliceraldeído/análogos & derivados , Propano/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antibacterianos/farmacologia , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Gliceraldeído/metabolismo , Gliceraldeído/farmacologia , Humanos , Lactobacillus reuteri/metabolismo , Organoides/efeitos dos fármacos , Organoides/microbiologia , Estresse Oxidativo/efeitos dos fármacos , Probióticos/metabolismo , Propano/metabolismo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento
11.
Cell Calcium ; 91: 102268, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32827867

RESUMO

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer's disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities. We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid ß protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection. Remarkably, our results indicate, as proof-of-principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Transportador 3 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Substâncias Protetoras/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Córtex Cerebral/patologia , Gliceraldeído , Humanos , Modelos Biológicos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia
12.
J Biol Phys ; 46(3): 283-295, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32617795

RESUMO

In the terrestrial dipolar magnetic field, magnetic circular dichroism (MCD) of UV sunlight by paramagnetic O2 in an Archean atmosphere (mostly CO2 and N2) results in circular polarization anisotropy (~ 10-10). This is used to calculate enantiomeric excess (EE~10-13) of glyceraldehyde (3-carbon sugar) with a model that includes racemic production and asymmetric photolysis of its enantiomers. The sign and magnitude of enantiomeric excess (EE) vary with the Earth's latitude. Unlike random noise fluctuation in spontaneous mirror symmetry breaking (SMSB) models, the sign of EE is deterministic and constant over large areas of prebiotic Earth. The magnitude is several orders greater than the mean amplitude of stochastically fluctuating EE. MCD could provide the initial EE for growth of homochirality by asymmetric autocatalysis.


Assuntos
Atmosfera , Dicroísmo Circular , Gliceraldeído/química , Fenômenos Magnéticos , Fotólise , Prebióticos/análise , Anisotropia , Estereoisomerismo
13.
Exp Eye Res ; 196: 108035, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353427

RESUMO

Axonal transport blockade is an initial step in retinal ganglion cell (RGC) degeneration in glaucoma and targeting maintenance of normal axonal transport could confer neuroprotection. We present an objective, quantitative method for assessing axonal transport blockade in mouse glaucoma models. Intraocular pressure (IOP) was elevated unilaterally in CD1 mice for 3 days using intracameral microbead injection. Longitudinal sections of optic nerve head (ONH) were immunofluorescently labeled for myelin basic protein (MBP) and amyloid precursor protein (APP), which is transported predominantly orthograde by neurons. The beginning of the myelin transition zone, visualized with the MBP label, was more posterior with elevated IOP, 288.8 ± 40.9 µm, compared to normotensive control eyes, 228.7 ± 32.7 µm (p = 0.030, N = 6 pairs). Glaucomatous regional APP accumulations in retina, prelaminar ONH, unmyelinated ONH, and myelinated optic nerve were identified by objective qualification of pixels with fluorescent intensity greater than the 97.5th percentile value of control eyes (suprathreshold pixels). This method segregated images with APP blockade from those with normal transport of APP. The fraction of suprathreshold pixels was significantly higher following IOP elevation than in normotensive controls in the unmyelinated ONH and myelinated nerve regions (paired analyses, p = 0.02 and 0.003, respectively, N = 12), but not in retina or prelaminar ONH (p = 0.91 and 0.08, respectively). The mean intensity of suprathreshold pixels was also significantly greater in glaucoma than in normotensive controls in prelaminar ONH, unmyelinated ONH and myelinated optic nerve (p = 0.01, 0.01, 0.002, respectively). Using this method, subconjunctival glyceraldehyde, which is known to worsen long-term RGC loss with IOP elevation, also produced greater APP blockade, but not statistically significant compared to glaucoma alone. Systemic losartan, which aids RGC axonal survival in glaucoma, reduced APP blockade, but not statistically significant compared to glaucoma alone. The method provides a short-term assessment of axonal injury for use in initial tests of neuroprotective therapies that may beneficially affect RGC transport in animal models of glaucoma.


Assuntos
Transporte Axonal/fisiologia , Modelos Animais de Doenças , Pressão Intraocular/fisiologia , Hipertensão Ocular/metabolismo , Disco Óptico/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anti-Hipertensivos/uso terapêutico , Axônios/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Gliceraldeído/uso terapêutico , Losartan/uso terapêutico , Camundongos , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Amielínicas/metabolismo , Nervo Óptico/metabolismo , Tonometria Ocular
14.
Bioorg Med Chem ; 28(10): 115464, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32249029

RESUMO

A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor d-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and d-glyceraldehyde was performed by l-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars d-sorbose and d-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldOS.coe) was expressed in E. coli and the purified AldOS.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldOS.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of d-sorbose and d-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.


Assuntos
Aldeído Liases/metabolismo , Gliceraldeído/metabolismo , Streptomyces coelicolor/enzimologia , Gliceraldeído/química , Glicerol/química , Glicerol/metabolismo , Conformação Molecular
15.
Int J Mol Sci ; 21(7)2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32283652

RESUMO

Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10-5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Compostos de Piridínio/farmacologia , Biomarcadores , Células Cultivadas , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Produtos Finais de Glicação Avançada/farmacologia , Gliceraldeído/análogos & derivados , Humanos , Túbulos Renais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Estresse Oxidativo/efeitos dos fármacos , Compostos de Piridínio/química , Espécies Reativas de Oxigênio/metabolismo
16.
Astrobiology ; 20(1): 1-14, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31977256

RESUMO

Extreme factors such as space microgravity, radiation, and magnetic field differ from those that occur on Earth. Microgravity may induce and select some microorganisms for physiological, metabolic, and/or genetic variations. This study was conducted to determine the effects of simulated microgravity conditions on the metabolism and gene expression of the probiotic bacterium Lactobacillus reuteri DSM17938. To investigate microbial response to simulated microgravity, two devices-the rotating wall vessel (RWV) and the random positioning machine (RPM)-were used. Microbial growth, reuterin production, and resistance to gastrointestinal passage were assessed, and morphological characteristics were analyzed by scanning electron microscopy. The expression of some selected genes that are responsive to stress conditions and to bile salts stress was evaluated through real-time quantitative polymerase chain reaction assay. Monitoring of bacterial growth, cell size, and shape under simulated microgravity did not reveal differences compared with 1 × g controls. On the contrary, an enhanced production of reuterin and a greater tolerance to the gastrointestinal passage were observed. Moreover, some stress genes were upregulated under RWV conditions, especially after 24 h of treatment, whereas RPM conditions seemed to determine a downregulation over time of the same stress genes. These results show that simulated microgravity could alter some physiological characteristics of L. reuteri DSM17938 with regard to tolerance toward stress conditions encountered on space missions and could be useful to elucidate the adaptation mechanisms of microbes to the space environment.


Assuntos
Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Gliceraldeído/análogos & derivados , Lactobacillus reuteri/crescimento & desenvolvimento , Lactobacillus reuteri/genética , Propano/metabolismo , Estresse Fisiológico/genética , Simulação de Ausência de Peso , Gliceraldeído/metabolismo , Lactobacillus reuteri/ultraestrutura
17.
Cell Prolif ; 53(2): e12702, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31628715

RESUMO

OBJECTIVES: Carnosine (ß-alanyl-l-histidine) is a naturally occurring dipeptide that selectively inhibits cancer cell growth, possibly by influencing glucose metabolism. As its precise mode of action and its primary targets are unknown, we analysed carnosine's effect on metabolites and pathways in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells, U87, T98G and LN229, were treated with carnosine, and metabolites were analysed by gas chromatography coupled with mass spectrometry. Furthermore, mitochondrial ATP production was determined by extracellular flux analysis and reaction products of carnosine were investigated using mass spectrometry. RESULTS: Carnosine decreased the intracellular abundance of several metabolites indicating a reduced activity of the pentose phosphate pathway, the malate-aspartate shuttle and the glycerol phosphate shuttle. Mitochondrial respiration was reduced in U87 and T98G but not in LN229 cells, independent of whether glucose or pyruvate was used as substrate. Finally, we demonstrate non-enzymatic reaction of carnosine with dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. However, glycolytic flux from glucose to l-lactate appeared not to be affected by the reaction of carnosine with the metabolites. CONCLUSIONS: Carnosine reacts non-enzymatically with glycolytic intermediates reducing the activity of the pentose phosphate pathway which is required for cell proliferation. Although the activity of the malate-aspartate and the glycerol phosphate shuttle appear to be affected, reduced mitochondrial ATP production under the influence of the dipeptide is cell-specific and appears to be independent of the effect on the shuttles.


Assuntos
Carnosina/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Via de Pentose Fosfato/fisiologia , Trifosfato de Adenosina/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Glucose/metabolismo , Gliceraldeído/metabolismo , Glicólise/fisiologia , Humanos , Malatos/metabolismo , Mitocôndrias/metabolismo , Fosfatos/metabolismo
18.
Cell Metab ; 31(1): 115-130.e6, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31708445

RESUMO

Iron is a central micronutrient needed by all living organisms. Competition for iron in the intestinal tract is essential for the maintenance of indigenous microbial populations and for host health. How symbiotic relationships between hosts and native microbes persist during times of iron limitation is unclear. Here, we demonstrate that indigenous bacteria possess an iron-dependent mechanism that inhibits host iron transport and storage. Using a high-throughput screen of microbial metabolites, we found that gut microbiota produce metabolites that suppress hypoxia-inducible factor 2α (HIF-2α) a master transcription factor of intestinal iron absorption and increase the iron-storage protein ferritin, resulting in decreased intestinal iron absorption by the host. We identified 1,3-diaminopropane (DAP) and reuterin as inhibitors of HIF-2α via inhibition of heterodimerization. DAP and reuterin effectively ameliorated systemic iron overload. This work provides evidence of intestine-microbiota metabolic crosstalk that is essential for systemic iron homeostasis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ferritinas/metabolismo , Microbioma Gastrointestinal , Ferro/metabolismo , Lactobacillus/metabolismo , Adolescente , Animais , Antibacterianos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diaminas/farmacologia , Dimerização , Duodeno/efeitos dos fármacos , Duodeno/microbiologia , Fezes/microbiologia , Feminino , Ferritinas/genética , Microbioma Gastrointestinal/fisiologia , Gliceraldeído/análogos & derivados , Gliceraldeído/farmacologia , Homeostase , Humanos , Lactobacillus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Organoides/efeitos dos fármacos , Organoides/microbiologia , Probióticos/farmacologia , Propano/farmacologia , Transdução de Sinais/efeitos dos fármacos
19.
Carbohydr Res ; 487: 107885, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31816468

RESUMO

A method for quantification of glyceraldehyde (GA), dihydroxyacetone (DHA) and glycerol (GLY) by gas chromatography coupled to a flame ionization detector (GC-FID) involving one-step derivatization into trimethylsilyl ethers is presented. In pyridine, DHA and GA showed predominant peaks assigned to dimeric structures and smaller peaks corresponding to the monomers. The later were identified by GC-MS as their completely derivatized molecules and were useful for construction of calibration curves with high linear correlation. On the other hand, DHA dimers were completely dissociated in water but GA dimers remained whereas with both, intermediates peaks arose which were associated to hydrated trymethyil silyl species. A calibration approach involving the sum of areas of most relevant peaks associated to aqueous solutions of GA and DHA was developed. Replicates measurements of a problem solution were in accordance with the results obtained by a well stablished HPLC technique. The coefficient of variation was below 5% for GLY and below 12% for GA and DHA. Compared with the HPLC method, the new GC-FID method presented a similar limit of quantification in the case of GA whereas for GLY and DHA a one-order-of-magnitude increase of sensitivity was achieved. TMS derivatives of GA and DHA without prior oximation enable a useful technique to study the equilibrium of the different tautomeric forms in solution.


Assuntos
Di-Hidroxiacetona/análise , Gliceraldeído/análise , Glicerol/análise , Calibragem , Cromatografia Gasosa , Estrutura Molecular
20.
Enzyme Microb Technol ; 133: 109456, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874684

RESUMO

Dihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.


Assuntos
Aldeído Liases/metabolismo , Escherichia coli/metabolismo , Cetoses/biossíntese , Biocatálise , Biotransformação , Frutose/metabolismo , Gliceraldeído/metabolismo , Glicerol/metabolismo , Microbiologia Industrial , Sorbose/metabolismo , Especificidade por Substrato
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