RESUMO
Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).
Assuntos
Glutarredoxinas , Hydra , Animais , Humanos , Glutarredoxinas/genética , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Hydra/genética , Hydra/metabolismo , Antioxidantes/metabolismo , Filogenia , Peróxido de Hidrogênio , Peixe-Zebra/metabolismo , Índia , Proteínas/química , Oxirredução , Glutationa/metabolismoRESUMO
Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.
Assuntos
Hydra , Tiorredoxina Dissulfeto Redutase , Animais , Humanos , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Hydra/genética , Hydra/metabolismo , Selenocisteína/química , Selenocisteína/metabolismo , Dissulfeto de Glutationa/metabolismo , Peróxido de Hidrogênio , Filogenia , Dinitroclorobenzeno , Células HEK293 , Glutationa/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Oxirredução , Antioxidantes/metabolismo , Mamíferos/metabolismoRESUMO
Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2 O2 ) is the most commonly used ROS as second messenger in the cell. It is a relatively long-lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury-induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2 O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2 O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2 O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of ß-catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2 O2 . Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.
Assuntos
Reparo do DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes Essenciais , Hydra/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Regeneração/efeitos dos fármacos , Animais , Hydra/fisiologiaAssuntos
Hydra , Animais , Hydra/genética , Glutationa Sintase , Sequência de Aminoácidos , Clonagem Molecular , GlutationaRESUMO
BACKGROUND: Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. METHODS: In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. RESULTS: In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500â¯J/m2) by 72â¯h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. CONCLUSIONS: Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Hydra/enzimologia , Raios Ultravioleta , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Hydra/genética , Hydra/efeitos da radiação , Filogenia , Homologia de Sequência , Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/genéticaRESUMO
BACKGROUND: Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. METHODS: Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. RESULTS: We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3ß by phosphorylation. Inactivated GSK-3ß leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). CONCLUSIONS: Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3ß, together resulting in activation of HO-1. GENERAL SIGNIFICANCE: We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc.
Assuntos
Antioxidantes/farmacologia , Diterpenos/farmacologia , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais , Morte Celular/efeitos dos fármacos , Heme Oxigenase-1/genética , Células Hep G2 , Humanos , Peróxido de Hidrogênio/toxicidade , Fator 2 Relacionado a NF-E2/genética , Regulação para CimaRESUMO
Geraniin is a hydrolysable tannin, widely present in many plant species, specifically used in traditional medicines. It has been shown to exhibit strong antioxidant activity in vitro. This study was performed to investigate hepatoprotective activity of geraniin against carbon tetrachloride (CCl4) induced damage in Swiss albino mice. Mice were treated with 30 and 60 mg/kg geraniin for 10 days followed by CCl4 administration for 24 h. Increase in Serum biochemical marker enzymes and histological deteriorative changes of liver tissue after CCl4 administration were attenuated by geraniin. Geraniin significantly reduced CCl4 induced lipid peroxidation, increase in amount of glutathione, glutathione reductase and Heme oxygenase-1 (HO-1). On the other hand it inhibited significant reduction in catalase activity and expression caused by CCl4 administration. Pre-treatment with geraniin reduced phosphorylation of translation initiation factor eIF2α, at serine 51, caused by CCl4 exposure and reduced elevated expression of its upstream kinase, Heme-regulated Inhibitor (HRI). These results clearly demonstrate hepatoprotective activity of geraniin against CCl4-induced acute hepatotoxicity via its free radical scavenging and antioxidant activities.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Glucosídeos/administração & dosagem , Heme Oxigenase-1/metabolismo , Taninos Hidrolisáveis/administração & dosagem , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Albinismo Oculocutâneo , Animais , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Resultado do TratamentoRESUMO
BACKGROUND: Diabetes mellitus is a metabolic disorder characterized by hyperglycaemia resulting from uncontrolled glucose regulation. Reactive oxygen species are recognized as one link between hyperglycaemia and diabetic complications. Studies have shown that diabetes mellitus is associated with decreases in antioxidant potential and increased formation of free radicals leading to oxidative stress. The present study was undertaken because an unequivocal demonstration that control of hyperglycaemia can reduce oxidative stress is still lacking. METHODS: In the present study, we investigated oxidative stress profile of normal, streptozotocin-induced diabetic, insulin-treated and untreated diabetic animals. On the one hand, oxidative damage caused to lipids, proteins and DNA was measured. On other hand, antioxidant defense was measured in terms of specific activities of antioxidant enzymes (AOEs) and antioxidant molecules. RESULTS: It was observed that the damage to lipids, proteins and DNA caused by free radicals increased in diabetic animals compared with that in controls. In diabetic animals not treated with insulin, damage to all biological molecules increased further significantly (p ≤ 0.005). Changes in AOEs from different tissues were complex depicting a varied AOE level in different tissues. Insulin treatment significantly improved the oxidative stress profile in all tissues studies. CONCLUSIONS: The control of hyperglycaemia improves oxidative stress profile, that is, the ability of cells to cope up with oxidative stress.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Hiperglicemia/fisiopatologia , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Glicemia/metabolismo , Dano ao DNA , Diabetes Mellitus Experimental/induzido quimicamente , Feminino , Técnicas In Vitro , Peroxidação de Lipídeos , Masculino , Camundongos , Oxirredução , Carbonilação Proteica/fisiologia , Espécies Reativas de Oxigênio/metabolismo , EstreptozocinaRESUMO
BACKGROUND: There exist several reports demonstrating enhancement in oxidative stress in diabetic patients; however, serial and comprehensive measurement of oxidative stress parameters in newly diagnosed diabetic patients is not yet reported. We measured the oxidative stress parameters in diabetic patients serially from the time of diagnosis and after starting treatment to study their association with glycaemia, insulin resistance and ß-cell function. METHODS: Fifty-four newly diagnosed diabetic patients were studied at diagnosis and 4 and 8 weeks after initiating anti-hyperglycaemic treatment. Oxidative stress parameters included activity of antioxidant enzymes, concentration of antioxidant molecules and damage markers. Oxidative stress score was computed as a collective measure of oxidative stress to interpret total oxidative stress state. Association of changing glucose levels with changing oxidative stress parameters over 8 weeks and association of oxidative stress score with insulin resistance and ß-cell function was analysed by homeostasis model assessment (HOMA-IR and HOMA-ß, respectively). RESULTS: Eight weeks of treatment improved HbA1C from 9.8 ± 2.1 to 7.7 ± 1.0%. There was a significant increase in oxidative stress in diabetic patients [23.8 (95% CI 20.0, 27.6)] compared with non-diabetic subjects [-1.2 (-3.4, 0.9)] (p < 0.001). Non-diabetic subjects showed a stable status over 8 weeks. Improvement in hyperglycaemia in diabetic patients was associated with an improvement in oxidative stress parameters irrespective of the anti-diabetic treatment received. Oxidative stress score fell after 8 weeks and was significantly associated with an improvement in HOMA-ß (standardized ß = -0.38, p < 0.01) but not with HOMA-IR. CONCLUSIONS: Controlling hyperglycaemia in diabetic patients alleviates oxidative stress within 8 weeks of treatment, and improvement in oxidative stress parameters was related to an improved ß-cell function.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/fisiologia , Estresse Oxidativo/fisiologia , Adulto , Antioxidantes/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Seguimentos , Hemoglobinas Glicadas/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/fisiopatologia , Hipoglicemiantes/farmacologia , Insulina/sangue , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Peróxidos Lipídicos/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Carbonilação Proteica/fisiologia , Estudos Retrospectivos , Superóxido Dismutase/metabolismo , Resultado do TratamentoRESUMO
Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.
Assuntos
Sequência de Aminoácidos , Glutarredoxinas , Hydra , Animais , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/química , Hydra/genética , Hydra/metabolismo , Hydra/enzimologia , Humanos , Clonagem Molecular , Domínios Proteicos , Filogenia , Proliferação de CélulasRESUMO
BACKGROUND: Pterostilbene, a methoxylated analog of Resveratrol, is gradually gaining more importance as a therapeutic drug owing to its higher lipophilicity, bioavailability and biological activity than Resveratrol. This study was undertaken to characterize its ability to scavenge free radicals such as superoxide, hydroxyl and hydrogen peroxide and to protect bio-molecules within a cell against oxidative insult. METHODS: Anti-oxidant activity of Pterostilbene was evaluated extensively by employing several in vitro radical scavenging/inhibiting assays and pulse radiolysis study. In addition, its ability to protect rat liver mitochondria against tertiary-butyl hydroperoxide (TBHP) and hydroxyl radical generated oxidative damage was determined by measuring the damage markers such as protein carbonyls, protein sulphydryls, lipid hydroperoxides, lipid peroxides and 8-hydroxy-2'-deoxyguanosine. Pterostilbene was also evaluated for its ability to inhibit â¢OH radical induced single strand breaks in pBR322 DNA. RESULT: Pterostilbene exhibited strong anti-oxidant activity against various free radicals such as DPPH, ABTS, hydroxyl, superoxide and hydrogen peroxide in a concentration dependent manner. Pterostilbene conferred protection to proteins, lipids and DNA in isolated mitochondrial fractions against TBHP and hydroxyl radical induced oxidative damage. It also protected pBR322 DNA against oxidative assault. CONCLUSIONS: Thus, present study provides an evidence for the strong anti-oxidant property of Pterostilbene, methoxylated analog of Resveratrol, thereby potentiating its role as an anti-oxidant.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Estilbenos/farmacologia , Animais , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Feminino , Radicais Livres/metabolismo , Fígado/química , Fígado/efeitos dos fármacos , Mitocôndrias/química , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The aim of this study was to check the effect of long-term oral glutathione (GSH) supplementation on alteration in gut microbiome of Indian diabetic individuals. Early morning fresh stool sample of diabetic individuals recruited in a randomized clinical trial wherein they were given 500 mg GSH supplementation orally once a day for a period of 6 months was collected and gut microbiome was analysed using high throughput 16S rRNA metagenomic sequencing. Long-term GSH supplementation as reported in our earlier work showed significant increase in body stores of GSH and stabilized decreased glycated haemoglobin (HbA1c). Analysis of gut microbiome revealed that abundance of phylum Proteobacteria significantly decreased (P < 0.05) in individuals with GSH supplementation after 6 months compared to those without it. Beneficial dominant genera such as Megasphaera, Bacteroides, and Megamonas were found to be significantly enriched (P < 0.05), while pathogenic Escherichia/Shigella was found to be depleted (P < 0.05) after supplementation. Data clearly demonstrate that GSH supplementation along with antidiabetic treatment helps restore the gut microbiome by enriching beneficial bacteria of healthy gut and reducing significantly the load of pathogenic bacteria of diabetic gut.
Assuntos
Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Humanos , RNA Ribossômico 16S/genética , Glutationa , Diabetes Mellitus Tipo 2/tratamento farmacológico , Suplementos NutricionaisRESUMO
Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.
Assuntos
Cnidários , Hydra , Animais , Clonagem Molecular , Cnidários/metabolismo , Humanos , Hydra/genética , Hydra/metabolismo , Peróxido de Hidrogênio , Índia , Oxirredução , Filogenia , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Since its discovery by Abraham Trembley in 1744, hydra has been a popular research organism. Features like spectacular regeneration capacity, peculiar tissue dynamics, continuous pattern formation, unique evolutionary position, and an apparent lack of organismal senescence make hydra an intriguing animal to study. While a large body of work has taken place, particularly in the domain of evolutionary developmental biology of hydra, in recent years, the focus has shifted to molecular mechanisms underlying various phenomena. DNA repair is a fundamental cellular process that helps to maintain integrity of the genome through multiple repair pathways found across taxa, from archaea to higher animals. DNA repair capacity and senescence are known to be closely associated, with mutations in several repair pathways leading to premature ageing phenotypes. Analysis of DNA repair in an animal like hydra could offer clues into several aspects including hydra's purported lack of organismal ageing, evolution of DNA repair systems in metazoa, and alternative functions of repair proteins. We review here the different DNA repair mechanisms known so far in hydra. Hydra genes from various DNA repair pathways show very high similarity with their vertebrate orthologues, indicating conservation at the level of sequence, structure, and function. Notably, most hydra repair genes are more similar to deuterostome counterparts than to common model invertebrates, hinting at ancient evolutionary origins of repair pathways and further highlighting the relevance of organisms like hydra as model systems. It appears that hydra has the full repertoire of DNA repair pathways, which are employed in stress as well as normal physiological conditions and may have a link with its observed lack of senescence. The close correspondence of hydra repair genes with higher vertebrates further demonstrates the need for deeper studies of various repair components, their interconnections, and functions in this early metazoan.
RESUMO
Nucleotide excision repair (NER) pathway is a DNA repair mechanism that rectifies a wide spectrum of DNA lesions. Xeroderma pigmentosum group of proteins (XPA through XPG) orchestrate the NER pathway in humans. We have earlier studied XPA homolog from Hydra (HyXPA) and found it to be similar to human XPA. Here, we examined if HyXPA can functionally complement human XPA-deficient cells and reduce their sensitivity to UV radiation. We found that HyXPA was able to partially rescue XPA-deficient human cells from UV by its binding to chromatin of UV-irradiated cells. However, HyXPA failed to bind replication protein A (RPA70), a key interacting partner of human XPA in NER pathway. This could be attributed to changes in certain amino acid residues that have occurred during evolution, leading to prevention of some interactions between Hydra and human proteins.
Assuntos
Cromatina/química , Reparo do DNA , DNA/genética , Evolução Molecular , Tolerância a Radiação/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Expressão Gênica , Teste de Complementação Genética , Humanos , Hydra , Plasmídeos/química , Plasmídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Type 2 diabetes (T2D) is a complex metabolic syndrome characterized by insulin dysfunction and abnormalities in glucose and lipid metabolism. The gut microbiome has been recently identified as an important factor for development of T2D. In this study, a total of 102 subjects were recruited, and we have looked at the gut microbiota of prediabetics (PreDMs) (n = 17), newly diagnosed diabetics (NewDMs) (n = 11), and diabetics on antidiabetic treatment (KnownDMs) (n = 39) and compared them with healthy nondiabetics (ND) (n = 35). Twenty-five different serum biomarkers were measured to assess the status of diabetes and their association with gut microbiota. Our analysis revealed nine different genera as differentially abundant in four study groups. Among them, Akkermansia, Blautia, and Ruminococcus were found to be significantly (P < 0.05) decreased, while Lactobacillus was increased in NewDMs compared to ND and recovered in KnownDMs. Akkermansia was inversely correlated with HbA1c and positively correlated with total antioxidants. Compared to ND, there was increased abundance of Megasphaera, Escherichia, and Acidaminococcus and decreased abundance of Sutterella in KnownDMs. Among many taxa known to act as community drivers during disease progression, we observed genus Sutterella as a common driver taxon among all diabetic groups. On the basis of the results of random forest analysis, we found that the genera Akkermansia and Sutterella and that the serum metabolites fasting glucose, HbA1c, methionine, and total antioxidants were highly discriminative factors among studied groups. Taken together, our data revealed that gut microbial diversity of NewDMs but not of PreDMs is significantly different from that of ND. Interestingly, after antidiabetic treatment, the microbial diversity of KnownDMs tends to recover toward that of ND.IMPORTANCE Gut microbiota is considered to play a role in disease progression, and previous studies have reported an association of microbiome dysbiosis with T2D. In this study, we have attempted to investigate gut microbiota of ND, PreDMs, NewDMs, and KnownDMs. We found that the genera Akkermansia and Blautia decreased significantly (P < 0.05) in treatment-naive diabetics and were restored in KnownDMs on antidiabetic treatment. To the best of our knowledge, comparative studies on shifts in the microbial community in individuals of different diabetic states are lacking. Understanding the transition of microbiota and its association with serum biomarkers in diabetics with different disease states may pave the way for new therapeutic approaches for T2D.
RESUMO
Radioprotective activity of pure compounds isolated from the plant Phyllanthus amarus was studied using rat liver mitochondria and pBR322 plasmid DNA as an in vitro model system. These compounds were ellagitannins namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, and flavonoids namely rutin, and quercetin 3-O-glucoside. The activity was then correlated with their hydroxyl and superoxide radical scavenging activity. Both ellagitannins and flavonoids effectively prevented lipid peroxidation and protein oxidation in mitochondria. The compounds also prevented radiation induced single strand breaks in pBR322 plasmid DNA. The radioprotective activity of ellagitannins and flavonoids could be due to their ability to scavenge different radicals more or less efficiently, relieving the oxidative stress. Protection conferred by flavonoids, rutin and quercetin 3-O-glucoside to rat liver mitochondria and plasmid pBR322 DNA from radiation induced damage was due to their strong hydroxyl radical scavenging activity. The inhibitory effect of ellagitannins on lipid peroxidation in liver mitochondria was due to their efficient superoxide radical scavenging ability. This is the first report about the radioprotective activity of pure ellagitannins from Phyllanthus amarus.
Assuntos
Flavonoides/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos da radiação , Fenóis/farmacologia , Phyllanthus/química , Extratos Vegetais/administração & dosagem , Tolerância a Radiação/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Polifenóis , Ratos , Ratos WistarRESUMO
Insulin resistance (IR) is known to precede onset of type 2 diabetes and increased oxidative stress appears to be a deleterious factor leading to IR. In this study, we evaluated ability of pterostilbene (PTS), a methoxylated analogue of resveratrol and a known antioxidant, to reverse palmitic acid (PA)-mediated IR in HepG2 cells. PTS prevented reactive oxygen species (ROS) formation and subsequent oxidative lipid damage by reducing the expression of NADPH oxidase 3 (NOX3) in PA treated HepG2 cells. Hepatic glucose production was used as a measure of IR and PTS reversed PA-mediated increase in hepatic glucose production by reducing expression of genes coding for gluconeogenic enzymes namely glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase (PC); and their transcription factors cAMP response element binding protein (CREB) and fork head class Box O (FOXO1) along with its coactivator peroxisome proliferator-activated receptor gamma co-activator-1 α (PGC1α). PTS reversed PA-mediated activation of c-Jun N-terminal kinase (JNK), which in turn altered insulin signalling pathway by phosphorylating IRS-1 at Ser 307, leading to inhibition of phosphorylation of Akt and GSK-3ß. PTS also reduced PA-mediated lipid accumulation by reducing expression of transcription factors SREBP1c and PPARα. SREBP1c activates genes involved in fatty acid and triglyceride synthesis while PPARα activates CPT1, a rate limiting enzyme for controlling entry and oxidation of fatty acids into mitochondria. PTS, however, did not influence PA uptake confirmed by using BODIPY-labelled fluorescent C16 fatty acid analogue. Thus, our data provides a possible mechanistic explanation for reversal of PA-mediated IR in HepG2 cells.
Assuntos
Resistência à Insulina/genética , Ácido Palmítico/efeitos adversos , Estilbenos/uso terapêutico , Triglicerídeos/metabolismo , Células Hep G2 , Humanos , Estresse Oxidativo , Estilbenos/farmacologiaRESUMO
Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3ß). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Glucosídeos/metabolismo , Heme Oxigenase-1/genética , Taninos Hidrolisáveis/metabolismo , Antioxidantes/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Citoproteção/genética , Citoproteção/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glucosídeos/farmacologia , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Heme Oxigenase-1/metabolismo , Células Hep G2/metabolismo , Humanos , Taninos Hidrolisáveis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Medicina Tradicional Chinesa , MicroRNAs/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Diabetes in India has distinct genetic, nutritional, developmental and socio-economic aspects; owing to the fact that changes in gut microbiota are associated with diabetes, we employed semiconductor-based sequencing to characterize gut microbiota of diabetic subjects from this region. We suggest consolidated dysbiosis of eubacterial, archaeal and eukaryotic components in the gut microbiota of newly diagnosed (New-DMs) and long-standing diabetic subjects (Known-DMs) compared to healthy subjects (NGTs). Increased abundance of phylum Firmicutes (p = 0.010) and Operational Taxonomic Units (OTUs) of Lactobacillus (p < 0.01) were observed in Known-DMs subjects along with the concomitant graded decrease in butyrate-producing bacterial families like Ruminococcaceae and Lachnospiraceae. Eukaryotes and fungi were the least affected components in these subjects but archaea, except Methanobrevibacter were significantly decreased in them. The two dominant archaea viz. Methanobrevibacater and Methanosphaera followed opposite trends in abundance from NGTs to Known-DMs subjects. There was a substantial reduction in eubacteria, with a noticeable decrease in Bacteroidetes phylum (p = 0.098) and an increased abundance of fungi in New-DMs subjects. Likewise, opportunistic fungal pathogens such as Aspergillus, Candida were found to be enriched in New-DMs subjects. Analysis of eubacterial interaction network revealed disease-state specific patterns of ecological interactions, suggesting the distinct behavior of individual components of eubacteria in response to the disease. PERMANOVA test indicated that the eubacterial component was associated with diabetes-related risk factors like high triglyceride (p = 0.05), low HDL (p = 0.03), and waist-to-hip ratio (p = 0.02). Metagenomic imputation of eubacteria depict deficiencies of various essential functions such as carbohydrate metabolism, amino acid metabolism etc. in New-DMs subjects. Results presented here shows that in diabetes, microbial dysbiosis may not be just limited to eubacteria. Due to the inter-linked metabolic interactions among the eubacteria, archaea and eukarya in the gut, it may extend into other two domains leading to trans-domain dysbiosis in microbiota. Our results thus contribute to and expand the identification of biomarkers in diabetes.