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1.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361015

RESUMO

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.


Assuntos
Adaptação Fisiológica , Flores/metabolismo , Redes Reguladoras de Genes , Nelumbo/genética , Mapas de Interação de Proteínas , Proteoma/metabolismo , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nelumbo/crescimento & desenvolvimento , Nelumbo/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/genética , Temperatura
2.
J Microbiol ; 59(9): 819-826, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382148

RESUMO

Phosphate solubilizing fungi (PSF) have been widely applied to dissolve insoluble phosphates (IPs). However, the PSF usually demonstrates a different phosphate solubilizing capacity for various IPs. This study explored the mechanisms of Aspergillus niger for the dissolution of ferric phosphate (FePO4, Fe-P), and tricalcium phosphate (Ca3[PO4]2, Ca-P) regarding the tricarboxylic acid (TCA) cycle. Aspergillus niger has higher phosphorus (P) content released from Ca-P, reached the maximum value of 861 mg/L after seven days of incubation, compared with the 169 mg/L from Fe-P. Oxalic acid promoted the release of P from Ca-P through the formation of calcium oxalate. The presence of Fe-P can stimulate A. niger to secrete large amounts of citric acid, confirmed by the enhancement of citrate synthase (CS) activity. However, citric acid only promotes 0.5% of P released from Fe-P. Meanwhile, although oxalic acid still dominates the release of P from Fe-P, its abundance was significantly declined. In contrast, oxalic acid also shows a higher P release ratio in Ca-P than citric acid, i.e., 36% vs. 22%. This study points to the future usage of A. niger to dissolve IPs in soil required to enhance oxalic acid secretion.


Assuntos
Aspergillus niger/metabolismo , Fosfatos de Cálcio/metabolismo , Compostos Férricos/metabolismo , Aspergillus niger/genética , Fosfatos de Cálcio/química , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Ácido Cítrico/metabolismo , Compostos Férricos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Oxálico/metabolismo , Solo/química
3.
Artigo em Inglês | MEDLINE | ID: mdl-34298180

RESUMO

In the last decade, there has been an increase in the study of the ecology of deep-sea organisms. One way to understand an organism's ecology is the study of its metabolism. According to literature, deep-sea sharks possess a lower anaerobic enzyme activity than their shallow-water counterparts, but no difference has been observed regarding their aerobic enzyme activities. These studies have suggested deep-sea sharks should be slow and listless swimmers. However, other studies based on video observations have revealed differences in cruise swimming speed between different species. The present study examined muscles of squaliform sharks, including both luminous and non-luminous species. We combined measurements of the relative amounts of red and white muscle with assays of enzymes that are used as markers for aerobic (citrate synthase, malate dehydrogenase) and anaerobic (lactate dehydrogenase) metabolism, searching for a relationship with cruising speeds. Non-luminous deep-sea species displayed lower aerobic enzyme activities but similar anaerobic enzyme activities than the benthic shallow-water counterpart (Squalus acanthias). Conversely, luminous Etmopteridae species were found to have similar aerobic enzyme activities to S. acanthias but displayed lower anaerobic enzyme activities. Analyses revealed that red muscle proportion and aerobic enzyme activities were positively related to the cruise swimming speed. In contrast, Dalatias licha, which swims at the slowest cruise swimming speed ever recorded, presented a very low aerobic metabolic phenotype (lower aerobic marker enzymes and less red muscle). Finally, the values obtained for white muscle proportion and anaerobic metabolic phenotype suggested a high burst capacity for D. licha and non-luminous sharks.


Assuntos
Citrato (si)-Sintase/metabolismo , Proteínas de Peixes/metabolismo , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Músculo Esquelético/metabolismo , Tubarões/metabolismo , Animais , Citrato (si)-Sintase/genética , Proteínas de Peixes/genética , L-Lactato Desidrogenase/genética , Malato Desidrogenase/genética , Músculo Esquelético/crescimento & desenvolvimento , Tubarões/genética , Tubarões/crescimento & desenvolvimento , Natação
4.
Nutrients ; 13(7)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202724

RESUMO

BACKGROUND: A low-sodium (LS) diet reduces blood pressure, contributing to the prevention of cardiovascular diseases. However, intense dietary sodium restriction impairs insulin sensitivity and worsens lipid profile. Considering the benefits of aerobic exercise training (AET), the effect of LS diet and AET in hepatic lipid content and gene expression was investigated in LDL receptor knockout (LDLr-KO) mice. METHODS: Twelve-week-old male LDLr-KO mice fed a normal sodium (NS) or LS diet were kept sedentary (S) or trained (T) for 90 days. Body mass, plasma lipids, insulin tolerance testing, hepatic triglyceride (TG) content, gene expression, and citrate synthase (CS) activity were determined. Results were compared by 2-way ANOVA and Tukey's post-test. RESULTS: Compared to NS, LS increased body mass and plasma TG, and impaired insulin sensitivity, which was prevented by AET. The LS-S group, but not the LS-T group, presented greater hepatic TG than the NS-S group. The LS diet increased the expression of genes related to insulin resistance (ApocIII, G6pc, Pck1) and reduced those involved in oxidative capacity (Prkaa1, Prkaa2, Ppara, Lipe) and lipoprotein assembly (Mttp). CONCLUSION: AET prevented the LS-diet-induced TG accumulation in the liver by improving insulin sensitivity and the expression of insulin-regulated genes and oxidative capacity.


Assuntos
Dieta Hipossódica/efeitos adversos , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Condicionamento Físico Animal/fisiologia , Receptores de LDL/deficiência , Animais , Peso Corporal , Citrato (si)-Sintase/metabolismo , Expressão Gênica , Lipídeos/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Sódio na Dieta/metabolismo , Triglicerídeos/metabolismo
5.
Toxicology ; 459: 152852, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34246718

RESUMO

Current cancer therapies are successfully increasing the lifespan of cancer patients. Nevertheless, cardiotoxicity is a serious chemotherapy-induced adverse side effect. Doxorubicin (DOX) and mitoxantrone (MTX) are cardiotoxic anticancer agents, whose toxicological mechanisms are still to be identified. This study focused on DOX and MTX's cardiac mitochondrial damage and their molecular mechanisms. As a hypothesis, we also sought to compare the cardiac modulation caused by 9 mg/kg of DOX or 6 mg/kg of MTX in young adult mice (3 months old) with old control mice (aged control, 18-20 months old) to determine if DOX- and MTX-induced damage had common links with the aging process. Cardiac homogenates and enriched mitochondrial fractions were prepared from treated and control animals and analyzed by immunoblotting and enzymatic assays. Enriched mitochondrial fractions were also characterized by mass spectrometry-based proteomics. Data obtained showed a decrease in mitochondrial density in young adults treated with DOX or MTX and aged control, as assessed by citrate synthase (CS) activity. Furthermore, aged control had increased expression of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and manganese superoxide dismutase (MnSOD). Regarding the enriched mitochondrial fractions, DOX and MTX led to downregulation of proteins related to oxidative phosphorylation, fatty acid oxidation, amino acid metabolic process, and tricarboxylic acid cycle. MTX had a greater impact on malate dehydrogenase (MDH2) and pyruvate dehydrogenase E1 component subunit α (PDHA1). No significant proteomic changes were observed in the enriched mitochondrial fractions of aged control when compared to young control. To conclude, DOX and MTX promoted changes in several mitochondrial-related proteins in young adult mice, but none resembling the aged phenotype.


Assuntos
Envelhecimento/efeitos dos fármacos , Antibióticos Antineoplásicos/toxicidade , Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitoxantrona/toxicidade , Proteoma/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Citrato (si)-Sintase/metabolismo , Masculino , Camundongos , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/patologia , Tamanho do Órgão/efeitos dos fármacos
6.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299193

RESUMO

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.


Assuntos
Citrato (si)-Sintase/metabolismo , Suplementos Nutricionais , Glicerol Quinase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Engenharia Metabólica/métodos , Yarrowia/fisiologia , Biomassa , Meios de Cultura , Ácidos Cetoglutáricos/isolamento & purificação
7.
Am J Physiol Endocrinol Metab ; 321(1): E80-E89, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34121449

RESUMO

Because patients with chronic obstructive pulmonary disease (COPD) are often physically inactive, it is still unclear whether the lower respiratory capacity in the locomotor muscles of these patients is due to cigarette smoking per se or is secondary to physical deconditioning. Accordingly, the purpose of this study was to examine mitochondrial alterations in the quadriceps muscle of 10 mice exposed to 8 mo of cigarette smoke, a sedentary mouse model of emphysema, and 9 control mice, using immunoblotting, spectrophotometry, and high-resolution respirometry in permeabilized muscle fibers. Mice exposed to smoke displayed a twofold increase in the oxidative stress marker, 4-HNE, (P < 0.05) compared with control mice. This was accompanied by significant decrease in protein expression of UCP3 (65%), ANT (58%), and mitochondrial complexes II-V (∼60%-75%). In contrast, maximal ADP-stimulated respiration with complex I and II substrates (CON: 23.6 ± 6.6 and SMO: 19.2 ± 8.2 ρM·mg-1·s-1) or octanoylcarnitine (CON: 21.8 ± 9.0 and SMO: 16.5 ± 6.6 ρM·mg-1·s-1) measured in permeabilized muscle fibers, as well as citrate synthase activity, were not significantly different between groups. Collectively, our findings revealed that sedentary mice exposed to cigarette smoke for 8 mo, which is typically associated with pulmonary inflammation and emphysema, exhibited a preserved mitochondrial respiratory capacity for various substrates, including fatty acid, in the skeletal muscle. However, the mitochondrial adaptations induced by cigarette smoke favored the development of chronic oxidative stress, which can indirectly contribute to augment the susceptibility to muscle fatigue and exercise intolerance.NEW & NOTEWORTHY It is unclear whether the exercise intolerance and skeletal muscle mitochondrial dysfunction observed in patients with COPD is due to cigarette smoke exposure, per se, or if they are secondary consequences to inactivity. Herein, while long-term exposure to cigarette smoke induces oxidative stress and an altered skeletal muscle phenotype, cigarette smoke does not directly contribute to mitochondrial dysfunction. With this evidence, we demonstrate the critical role of physical inactivity in cigarette smoke-related skeletal muscle dysfunction.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/ultraestrutura , Fumaça/efeitos adversos , Tabaco , Animais , Citrato (si)-Sintase/metabolismo , Modelos Animais de Doenças , Enfisema/patologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Estresse Oxidativo , Consumo de Oxigênio , Músculo Quadríceps/ultraestrutura , Comportamento Sedentário
8.
Methods Mol Biol ; 2277: 371-389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080163

RESUMO

In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.


Assuntos
Bioquímica/métodos , Leucócitos Mononucleares/citologia , Mitocôndrias/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/química , Citrato (si)-Sintase/análise , Citrato (si)-Sintase/metabolismo , Creatina Quinase/metabolismo , Humanos , Mitocôndrias/química , Oxirredutases/metabolismo , Fenótipo , Fluxo de Trabalho
9.
Methods Mol Biol ; 2276: 87-102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060034

RESUMO

Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD+-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/patologia , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
10.
Int J Biol Macromol ; 183: 213-221, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-33910038

RESUMO

Citrate synthase (CS) catalyzes the formation of citrate and coenzyme A from acetyl-CoA and oxaloacetate. CS exists in two forms: type I and type II. We determined the citrate-bound crystal structure of type II CS from the Hymenobacter sp. PAMC 26554 bacterium (HyCS; isolated from Antarctic lichen). Citrate molecules bound to a cleft between the large and small domains of HyCS. Structural comparison of HyCS with other type II CSs revealed that type II CSs have a highly conserved flexible hinge region (residues G264-P265 in HyCS), enabling correct positioning of active site residues. Notably, the catalytic His266 residue of HyCS interacted with Trp262 in the inactive (unliganded open) state of other type II CSs, whereas the His266 residue moved to the active site via a small-domain swing motion, interacting with the bound citrate in the closed conformation of HyCS. However, type I CSs lack this tryptophan residue and face-to-edge interactions. Thus, type II CSs might have a unique domain-motion control mechanism enabling a tight allosteric regulation. An activity assay using a W262A mutant showed a Hill coefficient of 2.4; thus, the interaction between Trp262 and His266 was closely related to the positive cooperative ligand binding of type II CS.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteroidetes/enzimologia , Citrato (si)-Sintase/metabolismo , Ácido Cítrico/metabolismo , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteroidetes/genética , Sítios de Ligação , Domínio Catalítico , Citrato (si)-Sintase/química , Citrato (si)-Sintase/genética , Coenzima A/metabolismo , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
11.
FASEB J ; 35(5): e21490, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33829547

RESUMO

Endotherms in cold regions improve heat-producing capacity when preparing for winter. We know comparatively little about how this change is fueled by seasonal adaptation in cellular respiration. Thus, we studied the changes of mitochondrial function in red blood cells in sympatric Coal (Periparus ater), Blue (Cyanistes caeruleus), and Great (Parus major) tits between autumn and winter. These species differ more than twofold in body mass and in several aspects of their foraging ecology and social dominance, which could require differential seasonal adaptation of energy expenditure. Coal and Great tits in particular upregulated the mitochondrial respiration rate and mitochondrial volume in winter. This was not directed toward ATP synthesis, instead reflecting increased uncoupling of electron transport from ATP production. Because uncoupling is exothermic, this increased heat-producing capacity at the sub-cellular level in winter. This previously unexplored the route of thermogenesis in birds should be addressed in future work.


Assuntos
Aclimatação , Metabolismo Energético , Eritrócitos/fisiologia , Mitocôndrias/fisiologia , Passeriformes/fisiologia , Estações do Ano , Termogênese , Animais , Citrato (si)-Sintase/metabolismo , Temperatura Alta
12.
Oxid Med Cell Longev ; 2021: 6643871, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33728024

RESUMO

Heart failure (HF) is a complex chronic clinical disease characterized by among others the damage of the mitochondrial network. The disruption of the mitochondrial quality control and the imbalance in fusion-fission processes lead to a lack of energy supply and, finally, to cell death. BGP-15 (O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic acid amidoxime dihydrochloride) is an insulin sensitizer molecule and has a cytoprotective effect in a wide variety of experimental models. In our recent work, we aimed to clarify the mitochondrial protective effects of BGP-15 in a hypertension-induced heart failure model and "in vitro." Spontaneously hypertensive rats (SHRs) received BGP-15 or placebo for 18 weeks. BGP-15 treatment preserved the normal mitochondrial ultrastructure and enhanced the mitochondrial fusion. Neonatal rat cardiomyocytes (NRCMs) were stressed by hydrogen-peroxide. BGP-15 treatment inhibited the mitochondrial fission processes, promoted mitochondrial fusion, maintained the integrity of the mitochondrial genome, and moreover enhanced the de novo biogenesis of the mitochondria. As a result of these effects, BGP-15 treatment also supports the maintenance of mitochondrial function through the preservation of the mitochondrial structure during hydrogen peroxide-induced oxidative stress as well as in an "in vivo" heart failure model. It offers the possibility, which pharmacological modulation of mitochondrial quality control under oxidative stress could be a novel therapeutic approach in heart failure.


Assuntos
Insuficiência Cardíaca/patologia , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Oximas/farmacologia , Piperidinas/farmacologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Citrato (si)-Sintase/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Mitocondrial/genética , Dinaminas/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Genoma Mitocondrial , Insuficiência Cardíaca/etiologia , Hipertensão/complicações , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Peptídeo Natriurético Encefálico/metabolismo , Biogênese de Organelas , Estresse Oxidativo/efeitos dos fármacos , Oximas/administração & dosagem , Oximas/química , Consumo de Oxigênio/efeitos dos fármacos , Piperidinas/administração & dosagem , Piperidinas/química , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
13.
Plant Sci ; 305: 110835, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33691969

RESUMO

Anthocyanins are important flavonoid pigments in plants. Malonyl CoA is an important intermediate in anthocyanin synthesis, and citrate, formed by citrate synthase (CS) catalysing oxaloacetate, is the precursor for the formation of malonyl-CoA. CS is composed of two isoforms, mitochondrial citrate synthase (mCS), a key enzyme of the tricarboxylic acid (TCA) cycle, and citrate synthase (CSY) localizated in microbodies in plants. However, no CS isoform involvement in anthocyanin synthesis has been reported. In this study, we identified the entire CS family in petunia (Petunia hybrida): PhmCS, PhCSY1 and PhCSY2. We obtained petunia plants silenced for the three genes. PhmCS silencing resulted in abnormal development of leaves and flowers. The contents of citrate and anthocyanins were significantly reduced in flowers in PhmCS-silenced plants. However, silencing of PhCSY1 and/or PhCSY2 did not cause a visible phenotype change in petunia. These results showed that PhmCS is involved in anthocyanin synthesis and the development of leaves and flowers, and that the citrate involved in anthocyanin synthesis mainly derived from mitochondria rather than microbodies in petunia.


Assuntos
Antocianinas/biossíntese , Antocianinas/genética , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Flores/enzimologia , Flores/genética , Petunia/enzimologia , Petunia/genética , Regulação da Expressão Gênica de Plantas , Genes Mitocondriais
14.
Sci Rep ; 11(1): 2167, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500513

RESUMO

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.


Assuntos
Atorvastatina/farmacologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/patologia , Mitocôndrias Musculares/metabolismo , Obesidade/patologia , Animais , Biomarcadores/metabolismo , Respiração Celular , Citrato (si)-Sintase/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Metaboloma , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Suínos , Porco Miniatura , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
15.
Int J Mol Sci ; 22(2)2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33435350

RESUMO

In 1937, Sir H. A Krebs first published the Citric Acid Cycle, a unidirectional cycle with carboxylic acids. The original concept of the Citric Acid Cycle from Krebs' 1953 Nobel Prize lecture illustrates the unidirectional degradation of lactic acid to water, carbon dioxide and hydrogen. Here, we add the heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex, connecting the original Citric Acid Cycle to the flow of energy and material. The heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex catalyses the first reaction of the Citric Acid Cycle, the oxidation of lactate to pyruvate, and thus secures the provision of pyruvic acid. In addition, we modify Krebs' original concept by feeding the cycle with oxaloacetic acid. Our concept enables the integration of anabolic processes and allows adaption of the organism to recover ATP faster.


Assuntos
Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Piruvato Carboxilase/metabolismo , Animais , Exercício Físico , Humanos , L-Lactato Desidrogenase/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fosforilação Oxidativa , Prótons , Simportadores/metabolismo
16.
Parasitol Res ; 120(3): 1025-1035, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33501586

RESUMO

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.


Assuntos
Galinhas/parasitologia , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Coccidiose/veterinária , Eimeria tenella/enzimologia , Doenças das Aves Domésticas/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Sequência de Bases , Western Blotting , Citrato (si)-Sintase/imunologia , Citrato (si)-Sintase/isolamento & purificação , Clonagem Molecular , Coccidiose/parasitologia , Eimeria tenella/genética , Eimeria tenella/fisiologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Soros Imunes/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Merozoítos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Nitrilas/farmacologia , Piranos/farmacologia , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Esporozoítos/enzimologia , Esporozoítos/imunologia , Triazinas/farmacologia
17.
Life Sci ; 268: 118936, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33421523

RESUMO

AIMS: During oxidative stress mitochondria become the main source of endogenous reactive oxygen species (ROS) production. In the present study, we aimed to clarify the effects of pharmacological PARP-1 inhibition on mitochondrial function and quality control processes. MAIN METHODS: L-2286, a quinazoline-derivative PARP inhibitor, protects against cardiovascular remodeling and heart failure by favorable modulation of signaling routes. We examined the effects of PARP-1 inhibition on mitochondrial quality control processes and function in vivo and in vitro. Spontaneously hypertensive rats (SHRs) were treated with L-2286 or placebo. In the in vitro model, 150 µM H2O2 stress was applied on neonatal rat cardiomyocytes (NRCM). KEY FINDINGS: PARP-inhibition prevented the development of left ventricular hypertrophy in SHRs. The interfibrillar mitochondrial network were less fragmented, the average mitochondrial size was bigger and showed higher cristae density compared to untreated SHRs. Dynamin related protein 1 (Drp1) translocation and therefore the fission of mitochondria was inhibited by L-2286 treatment. Moreover, L-2286 treatment increased the amount of fusion proteins (Opa1, Mfn2), thus preserving structural stability. PARP-inhibition also preserved the mitochondrial genome integrity. In addition, the mitochondrial biogenesis was also enhanced due to L-2286 treatment, leading to an overall increase in the ATP production and improvement in survival of stressed cells. SIGNIFICANCE: Our results suggest that the modulation of mitochondrial dynamics and biogenesis can be a promising therapeutical target in hypertension-induced myocardial remodeling and heart failure.


Assuntos
Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Células Cultivadas , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Eletrocardiografia , Glutationa/metabolismo , Hipertrofia Ventricular Esquerda/etiologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/ultraestrutura , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/patologia , Peptídeo Natriurético Encefálico/sangue , Piperidinas/farmacologia , Quinazolinas/farmacologia , Ratos Endogâmicos SHR , Ratos Wistar
18.
J Sci Med Sport ; 24(3): 291-296, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32998848

RESUMO

OBJECTIVES: Optimized concurrent training regimes are warranted in physical training of military-, law enforcement- and rescue-personnel. This study investigated if four 15-min endurance training sessions weekly improve aerobic capacity and performance more than one 60-min endurance session weekly during the initial phase of a Basic Military Training program. DESIGN: A randomized training intervention study with functional and physiological tests before and after the intervention. METHODS: Military conscripts (n=290) were randomly allocated to three groups completing 9 weeks training. Weekly training consisted of four endurance and four strength training sessions lasting 15min each ('Micro-training': MIC); one strength and one endurance session lasting 60min each ('Classical-training': CLA) or two 60min sessions of standard military training ('Control-training': CON). RESULTS: Both 12-min (∼7-10%) and shuttle run performance (∼35-42%) improved (P≤0.001) similarly in all groups. Likewise, functional 2-min maximal repetition exercise capacity increased (P≤0.05) similarly in all groups (Lunges ∼17-24 %; PushUp ∼10-20%; AbdominalFlexions∼21-23%). Peak oxygen uptake changes depended on group (P≤0.05) with increases (P≤0.01) in MIC (7±7%, n=23) and CON (12±18%, n=17) and no changes in CLA. Maximal m. vastus lateralis citrate synthase activity decreased 14±26% (P≤0.001, n=18) in CLA. Likewise, maximal m. vastus lateralis 3-hydroxyacyl-CoA dehydrogenase activity decreased 8±17% in MIC (n=28) and 14±24% in CLA (n=18). CONCLUSIONS: Four 15-min endurance training sessions weekly improves running performance and strength-endurance similarly to one 60min session. Peak oxygen uptake only increases with more than one endurance session weekly and leg muscle oxidative capacity appears reduced after basic military training.


Assuntos
Treino Aeróbico/métodos , Tolerância ao Exercício/fisiologia , Militares , Treinamento de Força/métodos , Corrida/fisiologia , 3-Hidroxiacil-CoA Desidrogenase/metabolismo , Colesterol/sangue , Citrato (si)-Sintase/metabolismo , Feminino , Humanos , Masculino , Consumo de Oxigênio/fisiologia , Músculo Quadríceps/metabolismo , Fatores de Tempo
19.
J Sci Med Sport ; 24(5): 494-498, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33160857

RESUMO

OBJECTIVES: Measurements of protein content, enzymatic activity, and/or capillarization are frequently utilized as markers of skeletal muscle adaptation following exercise training. Whether changes in these markers of muscle adaptation are repeatable when individuals are repeatedly exposed to the same training stimulus is unknown. The purpose of this study was to test the repeatability of skeletal muscle adaptations to two identical training periods. METHODS: Ten active young males (age: 22 ±â€¯2 years; VO2max: 57 ±â€¯7 ml/kg/min) were exposed to two identical four-week periods of supervised high-intensity interval running (4 × 4 min at 90-95% of HRmax interspersed with 3-min at 70-75% HRmax) separated by a 3-month wash-out period. Vastus lateralis biopsies were obtained before and after each training period for the measurement of protein content, enzyme activity, and capillary density. RESULTS: Training-induced changes in citrate synthase (CS) maximal activity, protein content (PGC-1α, OXPHOS, and LDH-A), and capillary density were not repeatable within individuals (r = -0.52-0.15; ICCs: -0.42-0.04; CVs: 11-67%). Several OXPHOS complex subunits also demonstrated dissimilar group-level adaptations (period × time interaction effects, p < 0.05) with large differences (ηp2 > 0.4) between training periods. A large (ηp2 = 0.65) increase in capillary density was apparent irrespective of training period (main effect of time, p = 0.05). CONCLUSIONS: An individual (or a group of individuals) may exhibit dissimilar skeletal muscle adaptations when re-exposed to the same training stimulus. Our findings challenge the utility of classifying of individuals as high/low responders using measurements of mitochondrial protein content, CS activity and/or capillary density following a single training period.


Assuntos
Adaptação Fisiológica , Exercício Físico/fisiologia , Músculo Quadríceps/fisiologia , Corrida/fisiologia , Capilares/metabolismo , Citrato (si)-Sintase/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/metabolismo , Adulto Jovem
20.
Sci Rep ; 10(1): 19686, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184414

RESUMO

Sodium glucose transporter (SGLT)-2 inhibitors have consistently shown cardioprotective effects independent of the glycemic status of treated patients. In this study we aimed to investigate underlying mechanisms of short-term empagliflozin treatment in a mouse model of type II diabetes. Male db/db mice were fed a western type diet with or without enrichment with empagliflozin for 7 days. While glucose tolerance was significantly improved in empagliflozin treated mice, body weight and fasting insulin levels were comparable in both groups. Cardiac insulin signaling activity indicated by reduced proteinkinase B (AKT) phosphorylation was significantly decreased in the empagliflozin treated group. Remarkably, mitochondrial mass estimated by citrate synthase activity was significantly elevated in empagliflozin treated mice. Accordingly, mitochondrial morphology was significantly altered upon treatment with empagliflozin as analysed by transmission electron microscopy. Additionally, short-term empagliflozin therapy was associated with a changed cardiac tissue cytokine expression in favor of an anti-inflammatory pattern. Our data suggest that early cardioprotection in empagliflozin treated mice is independent of a reduction in body weight or hyperinsulinemia. Ameliorated mitochondrial ultrastructure, attenuated cardiac insulin signaling and diminished cardiac inflammation might contribute to the cardioprotective effects of empagliflozin.


Assuntos
Compostos Benzidrílicos/administração & dosagem , Cardiotônicos/administração & dosagem , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dieta Ocidental/efeitos adversos , Glucosídeos/administração & dosagem , Animais , Compostos Benzidrílicos/farmacologia , Peso Corporal/efeitos dos fármacos , Cardiotônicos/farmacologia , Citrato (si)-Sintase/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Masculino , Camundongos , Miocárdio/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resultado do Tratamento
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