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We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, â³UA) residue at their non-reducing end. The major "even-numbered" series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor "odd-numbered" series oligosaccharides carried a terminal reducing glucuronic acid residue instead. Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L-1 chondroitin or chondroitin oligosaccharides. All products were purified and fully characterized using NMR and mass spectrometry analyses. This is the first report of the microbial production of large chondro-oligosaccharides.
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A myocardial infarction can cause irreversible damage to the heart muscle. A promising approach for the treatment of myocardial infarction and prevention of severe complications is the application of cardiac patches or epicardial restraint devices. The challenge for the fabrication of cardiac patches is the replication of the fibrillar structure of the myocardium, in particular its anisotropy and local elasticity. In this study, we developed a chitosan-gelatin-guar gum-based biomaterial ink that was fabricated using 3D printing to create patterned anisotropic membranes. The experimental results were then used to develop a numerical model able to predict the elastic properties of additional geometries with tunable elasticity that could easily match the mechanical properties of the heart tissue (particularly the myocardium).
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Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air-liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. OBJECTIVES: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. METHODS: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. RESULTS: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 µL of 30 mg/mL TiO2 and 114 µL of 15 mg/mL TiO2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 µL.
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During intraocular lens (IOL) implantation it is not uncommon for the injector's nozzle-tip to get damaged. However, the damage has not been systematically described or evaluated using an objective scale. In this study we developed our own system-the Heidelberg Score for IOL Injector Damage ("HeiScore"), which was used to grade 60 injectors from four generations of injector models (Monarch III D, AcrySert C, UltraSert, AutonoMe) made by the same manufacturer. (Alcon Laboratories Inc.) HeiScore has six grades of nozzle-tip damage: no damage (which was graded 0); slight scratches (1), deep scratches (2), extensions (3), cracks (4) and bursts (graded number 5). The score for each injector model was the sum of all grades (total number), and we could compare the four injector models. The injectors showed varying damage profiles, from "no damage" to "crack". A tendency of a lower damage score in the newer generations of IOL injectors was noted. However, a statistically significant difference was observed only between Monarch III D and AutonoMe. The "Heidelberg Score for IOL Injector Damage" could efficiently and effectively evaluate the damage to IOL injector systems, which might help manufacturers optimize the positioning of the IOL in the injector during pre-loading.
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Implante de Lente Intraocular/instrumentação , Instrumentos Cirúrgicos/normas , Injeções/instrumentação , Fenômenos MecânicosRESUMO
Impaired glucose metabolism is observed in obesity and type 2 diabetes. Glucose controls gene expression through the transcription factor ChREBP in liver and adipose tissues. Mlxipl encodes 2 isoforms: ChREBPα, the full-length form (translocation into the nucleus is under the control of glucose), and ChREBPß, a constitutively nuclear shorter form. ChREBPß gene expression in white adipose tissue is strongly associated with insulin sensitivity. Here, we investigated the consequences of ChREBPß deficiency on insulin action and energy balance. ChREBPß-deficient male and female C57BL6/J and FVB/N mice were produced using CRISPR/Cas9-mediated gene editing. Unlike global ChREBP deficiency, lack of ChREBPß showed modest effects on gene expression in adipose tissues and the liver, with variations chiefly observed in brown adipose tissue. In mice fed chow and 2 types of high-fat diets, lack of ChREBPß had moderate effects on body composition and insulin sensitivity. At thermoneutrality, ChREBPß deficiency did not prevent the whitening of brown adipose tissue previously reported in total ChREBP-KO mice. These findings revealed that ChREBPß is dispensable for metabolic adaptations to nutritional and thermic challenges.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Glicemia/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/genética , Metabolismo Energético/genética , Regulação da Expressão Gênica , RNA/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large-scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption. Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed. A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100%. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP-free platform that could be adaptable for production of other biomolecules. KEY POINTS: ⢠A biomolecule production platform based on B. subtilis 3NA strain and a synthetic medium was tested for hyaluronic acid biosynthesis ⢠A fermentation process with the maximum volumetric productivity was designed ⢠A techno-economic analysis forecasts a significant reduction in the manufacturing cost compared to the current methods.
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Bacillus subtilis , Ácido Hialurônico , Animais , Bacillus subtilis/genética , Meios de Cultura , Fermentação , StreptococcusRESUMO
In 2021, the 100th anniversary of the isolation of insulin and the rescue of a child with type 1 diabetes from death will be marked. In this review, we highlight advances since the ingenious work of the four discoverers, Frederick Grant Banting, John James Rickard Macleod, James Bertram Collip and Charles Herbert Best. Macleoad closed his Nobel Lecture speech by raising the question of the mechanism of insulin action in the body. This challenge attracted many investigators, and the question remained unanswered until the third part of the 20th century. We summarize what has been learned, from the discovery of cell surface receptors, insulin action, and clearance, to network and precision medicine.
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Insulina , Descoberta do Conhecimento , Animais , Diabetes Mellitus Tipo 1 , Endocitose , História do Século XX , Humanos , Insulina/fisiologia , Descoberta do Conhecimento/história , Mapas de Interação de Proteínas , Receptor de Insulina/metabolismo , PesquisadoresRESUMO
Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated ß3N-acetylgalactosamine (GalNAc)-ß4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose ß-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated ß3-galactose (Gal)-ß4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.
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Condroitina/síntese química , Escherichia coli/enzimologia , Galactose/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Acetilglucosamina/metabolismo , Reatores Biológicos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Condroitina/química , Lactose/metabolismo , Engenharia Metabólica , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
nvestigations on adverse biological effects of nanoparticles (NP) are performed usually either in vivo on rodents or in vitro under submerged conditions where NP are in suspension into cell culture media. However, sedimentation of NP in vitro is a continuous process and to assess the exact deposited cellular dose remains difficult, as the cellular internal dose is a function of time. Moreover, the cellular responses to NP under submerged culture conditions or by exposing rodents by nose-only to NP aerosols might differ from those observed at physiological settings at the air-liquid interface (ALI). Rat alveolar NR8383 macrophages were exposed to aerosols at the air-liquid interface. We studied TiO2 NM105, a mixture of anatase and rutile. NR8383 cells were exposed to a single dose of 3.0 cm2/cm2 of TiO2 aerosol. Following RNA extraction, transcriptome allowing full coverage of the rat genome was performed, and differentially expressed genes were retrieved. Their products were analyzed for functions and interaction with String DB. Only 126 genes were differentially expressed and 98 were recognized by String DB and give us the gene expression signature of exposed rat alveolar NR8383 macrophages. Among them, 13 display relationships at a high confidence level and the ten most differentially expressed compared to unexposed cells were: Chac1, Ccl4, Zfp668, Fam129b, Nab2, Txnip, Id1, Cdc42ep3, Dusp6 and Myc, ranked from the most overexpressed to the most under-expressed. Some of them were previously described as over or under-expressed in NP exposed cell systems. We validated in our laboratory an easy-to-use device and a physiological relevant paradigm for studying the effects of cell exposure to TiO2. Ccl4 gene expression seems to be a positive marker of exposure evidenced as well as in vivo or in both in vitro conditions.
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Nanopartículas/toxicidade , Titânio/toxicidade , Aerossóis/toxicidade , Animais , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ratos , Suspensões/toxicidade , Transcriptoma/efeitos dos fármacosRESUMO
There are many studies concerning titanium dioxide (TiO2) nanoparticles (NP) toxicity. Nevertheless, there are few publications comparing in vitro and in vivo exposure, and even less comparing air-liquid interface exposure (ALI) with other in vitro and in vivo exposures. The identification and validation of common markers under different exposure conditions are relevant for the development of smart and quick nanotoxicity tests. In this work, cell viability was assessed in vitro by WST-1 and LDH assays after the exposure of NR8383 cells to TiO2 NP sample. To evaluate in vitro gene expression profile, NR8383 cells were exposed to TiO2 NP during 4 h at 3 cm2 of TiO2 NP/cm2 of cells or 19 µg/mL, in two settings-submerged cultures and ALI. For the in vivo study, Fischer 344 rats were exposed by inhalation to a nanostructured aerosol at a concentration of 10 mg/m3, 6 h/day, 5 days/week for 4 weeks. This was followed immediately by gene expression analysis. The results showed a low cytotoxic potential of TiO2 NP on NR8383 cells. Despite the absence of toxicity at the doses studied, the different exposures to TiO2 NP induce 18 common differentially expressed genes (DEG) which are involved in mitosis regulation, cell proliferation and apoptosis and inflammation transport of membrane proteins. Among these genes, we noticed the upregulation of Ccl4, Osm, Ccl7 and Bcl3 genes which could be suggested as early response biomarkers after exposure to TiO2 NP. On the other hand, the comparison of the three models helped us to validate the alternative ones, namely submerged and ALI approaches.
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Nanopartículas/toxicidade , Titânio/toxicidade , Administração por Inalação , Aerossóis/toxicidade , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inflamação , Masculino , Proteínas de Membrana/metabolismo , Mitose/efeitos dos fármacos , Nanoestruturas/toxicidade , Ratos , Ratos Endogâmicos F344 , Transcriptoma/efeitos dos fármacosRESUMO
Functionalized multi-walled carbon nanotubes (MWCNT) have become the focus of increased research interest, particularly in their application as tools in different areas, such as the biomedical field. Despite the benefits associated with functionalization of MWCNT, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display different toxicity profiles. For this study, we aim to compare NR8383 cells responses to three well-characterized MWCNT with varying functional groups. This study employed cytotoxicity assays, transcriptomics and proteomics to assess their toxicity using NR8383 rat alveolar macrophages as an in vitro model. The study findings indicated that all MWCNT altered ribosomal protein translation, cytoskeleton arrangement and induced pro-inflammatory response. Only functionalized MWCNT alter mTOR signaling pathway in conjunction with increased Lamtor gene expression. Furthermore, the type of functionalization was also important, with cationic MWCNT activating the transcription factor EB and inducing autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study proposes that MWCNT toxicity mechanisms are functionalization dependent and provides evidence that inflammatory response is a key event of carbon nanotubes toxicity.
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Perfilação da Expressão Gênica , Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , Animais , Autofagia , Cátions , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Expressão Gênica , L-Lactato Desidrogenase/metabolismo , Macrófagos Alveolares/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Nanoestruturas/química , Tamanho da Partícula , Proteômica , Ratos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Metal oxide nanoparticles (NPs), such as ZnO, ZnFe2O4, and Fe2O3, are widely used in industry. However, little is known about the cellular pathways involved in their potential toxicity. Here, we particularly investigated the key molecular pathways that are switched on after exposure to sub-toxic doses of ZnO, ZnFe2O4, and Fe2O3 in the in vitro rat alveolar macrophages (NR8383). As in our model, the calculated IC50 were respectively 16, 68, and more than 200 µg/mL for ZnO, ZnFe2O4, and Fe2O3; global gene and protein expression profiles were only analyzed after exposure to ZnO and ZnFe2O4 NPs. Using a rat genome microarray technology, we found that 985 and 1209 genes were significantly differentially expressed in NR8383 upon 4 h exposure to » IC50 of ZnO and ZnFe2O4 NPs, respectively. It is noteworthy that metallothioneins were overexpressed genes following exposure to both NPs. Moreover, Ingenuity Pathway Analysis revealed that the top canonical pathway disturbed in NR8383 exposed to ZnO and ZnFe2O4 NPs was eIF2 signaling involved in protein homeostasis. Quantitative mass spectrometry approach performed from both NR8383 cell extracts and culture supernatant indicated that 348 and 795 proteins were differentially expressed upon 24 h exposure to » IC50 of ZnO and ZnFe2O4 NPs, respectively. Bioinformatics analysis revealed that the top canonical pathways disturbed in NR8383 were involved in protein homeostasis and cholesterol biosynthesis for both exposure conditions. While VEGF signaling was specific to ZnO exposure, iron homeostasis signaling pathway was specific to ZnFe2O4 NPs. Overall, the study provides resource of transcriptional and proteomic markers of response to ZnO and ZnFe2O4 NP-induced toxicity through combined transcriptomics, proteomics, and bioinformatics approaches.
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Macrófagos Alveolares/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Animais , Linhagem Celular , Biologia Computacional/métodos , Homeostase , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Nanopartículas Magnéticas de Óxido de Ferro/toxicidade , Nanopartículas Metálicas/toxicidade , Nanopartículas/química , Nanopartículas/toxicidade , Proteômica/métodos , Ratos , Transdução de Sinais , Óxido de Zinco/química , Óxido de Zinco/toxicidadeRESUMO
Unfortunately, the author names in the author group section were incorrectly captured in the published online paper.
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Various amino acid (AA) metabolites are used as supplements to facilitate metabolic control and enhance responsiveness of insulin-sensitive tissues. ß-hydroxy-ß-methylbutyrate (HMB) is a leucine metabolite proposed to prevent muscle wasting and to mitigate insulin resistance. Taurine, commonly added to energizing drinks, is a metabolite of methionine and cysteine present in bile juice, and proposed to be involved in lipid digestion and to be pro-lipolytic in adipocytes. N-methyltyramine (NMT) is a phenylalanine metabolite found in orange juices at 0.1-3 ppm while its effects on lipid mobilization remain controversial. Here, the putative lipolytic effects of these AA metabolites were studied and it was tested whether they could enhance insulin antilipolytic response in adipocytes. Release of glycerol and non-esterified fatty acids (NEFAs) was measured after a 2-h incubation of adipocytes obtained from control and diet-induced obese mice or from obese patients. In mouse, none of the tested AA derivatives was lipolytic from 1 µM to 1 mM. These compounds did not improve insulin antilipolytic effect or isoprenaline lipolytic action, except for 1 mM NMT that impaired triacylglycerol breakdown in obese mice. In human adipocytes, HMB and taurine were not lipolytic, while NMT weakly activated glycerol and NEFA release at 1 mM. However, 100 µM NMT impaired isoprenaline-stimulated lipolysis in a manner that was hardly added to insulin antilipolytic effect. Since none of these AA derivatives acutely helped or replaced insulin antilipolytic effect in adipocytes, the present in vitro observations do not support their proposed insulin-sensitizing properties. Moreover, NMT, HMB, and taurine were not notably lipolytic.
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Adipócitos , Insulina/metabolismo , Lipólise/efeitos dos fármacos , Taurina/farmacologia , Tiramina/análogos & derivados , Valeratos/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adulto , Animais , Feminino , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Obesidade/metabolismo , Tiramina/farmacologiaRESUMO
Human exposure to airborne carbon nanotubes (CNT) is increasing because of their applications in different sectors; therefore, they constitute a biological hazard. Consequently, developing studies on CNT toxicity become a necessity. CNTs can have different properties in term of length, size and charge. Here, we compared the cellular effect of multiwall (MWCNTs) and single wall CNTs (SWCNTs). MWCNTs consist of multiple layers of graphene, while SWCNTs are monolayers. The effects of MWCNTs and SWCNTs were evaluated by the water-soluble tetrazolium salt cell proliferation assay on NR8383 cells, rat alveolar macrophage cell line (NR8383). After 24 hours of exposure, MWCNTs showed higher toxicity (50% inhibitory concentration [IC50 ] = 3.2 cm2 /cm2 ) than SWCNTs (IC50 = 44 cm2 /cm2 ). Only SWCNTs have induced NR8383 cells apoptosis as assayed by flow cytometry using the annexin V/IP staining test. The expression of genes involved in oxidative burst (Ncf1), inflammation (Nfκb, Tnf-α, Il-6 and Il-1ß), mitochondrial damage (Opa) and apoptotic balance (Pdcd4, Bcl-2 and Casp-8) was determined. We found that MWCNT exposure predominantly induce inflammation, while SWCNTs induce apoptosis and impaired mitochondrial function. Our results clearly suggest that MWCNTs are ideal candidates for acute inflammation induction. In vivo studies are required to confirm this hypothesis. However, we conclude that toxicity of CNTs is dependent on their physical and chemical characteristics.
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Poluentes Atmosféricos/toxicidade , Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Poluentes Atmosféricos/química , Animais , Linhagem Celular , Nanotubos de Carbono/química , Tamanho da Partícula , Ratos , Propriedades de SuperfícieRESUMO
Coenzyme Q (Q) is a redox lipid that is central for the energetic metabolism of eukaryotes. The biosynthesis of Q from the aromatic precursor 4-hydroxybenzoic acid (4-HB) is understood fairly well. However, biosynthetic details of how 4-HB is produced from tyrosine remain elusive. Here, we provide key insights into this long-standing biosynthetic problem by uncovering molecular details of the first and last reactions of the pathway in the yeast Saccharomyces cerevisiae, namely the deamination of tyrosine to 4-hydroxyphenylpyruvate by Aro8 and Aro9, and the oxidation of 4-hydroxybenzaldehyde to 4-HB by Hfd1. Inactivation of the HFD1 gene in yeast resulted in Q deficiency, which was rescued by the human enzyme ALDH3A1. This suggests that a similar pathway operates in animals, including humans, and led us to propose that patients with genetically unassigned Q deficiency should be screened for mutations in aldehyde dehydrogenase genes, especially ALDH3A1.
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Aldeído Desidrogenase/metabolismo , Vias Biossintéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona/metabolismo , Aldeído Desidrogenase/genética , Benzaldeídos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Humanos , Oxirredução , Parabenos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tirosina/genética , Tirosina/metabolismo , Ubiquinona/genéticaRESUMO
The heparosan synthase of Escherichia coli K5 is composed of the glycosyltransferases KfiA and KfiC which synthesize the polysaccharide heparosan (N-acetylheparosan). A third protein, KfiB, is required to stabilize the KfiAC complex in the bacteria and to transport this complex to the inner membrane where the initiation of polymerization occurs. In this report, we fused KfiC with the E. coli trigger factor (TF) to stabilize KfiC, thus activating the enzyme in the absence of KfiB. Different recombinant plasmids were constructed to compare the impact of the presence or absence of KfiB and the presence of the trigger factor as a fusion protein. Several E. coli BL21-derived strains were transformed with recombinant plasmids and cultivated in fed-batch conditions on minimal medium. The bTCA strain overexpressing fused TF-KfiC together with KfiA and KfiD, but lacking KfiB produced 1.5 g/L of total heparosan after 24 h of fed-batch cultivation. This heparosan was essentially intracellular early in the culture, providing evidence that KfiB primarily plays a role in the exportation process. However, over time, heparosan became mostly extracellular, likely due to passive diffusion or partial cell disruption upon product accumulation.
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Dissacarídeos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Expressão Gênica , Glicosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Escherichia coli/genética , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The classification of outdoor air pollution as carcinogenic for humans strengthens the increasing concern about particulate matter (PM). We previously demonstrated that PM exposure produces an antiapoptotic effect resulting from polycyclic aromatic hydrocarbons (PAH) and water-soluble components. In this study, we investigated transition metallic compounds, particularly iron, in order to decipher their underlying molecular mechanisms that prevent apoptosis. Human bronchial epithelial cells were exposed for 4 h to different PM samples with established antiapoptotic effect (e.g. PM-AW) or not (e.g. PM-VS) or to their metallic components (Fe, Mn, Zn and Al) before apoptosis induction by the calcium ionophore A23187 or Staurosporine. PM-AW, Fe, Mn and Al significantly reduced induced apoptosis. The antiapoptotic effect of Fe was enhanced by benzo(a)pyrene, a typical PAH compound, but was totally reversed by the iron chelator, deferiprone. Furthermore, particles and iron triggered cellular ROS generation and prevented the depletion in glutathione levels observed during A23187-induced apoptosis. In contrast to benzo(a)pyrene, PM-AW and Fe rapidly activated NRF2, subsequently upregulated several target genes (HO1, NQO1 and GPX1) and modulated some genes which control cell death (BCL2, BAX and p53). The key role of the NRF2 pathway in the antiapoptotic effect mediated by Fe and PM was demonstrated using siRNA technology. Our results suggest that the iron component participates in the antiapoptotic effect of PM by activating a NRF2-dependent antioxidant process. As resisting to cell death is one of the hallmarks of cancer cells, these findings provide additional clues for understanding the development of lung cancer after atmospheric pollution exposure.
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Apoptose/efeitos dos fármacos , Ferro/toxicidade , Neoplasias Pulmonares/etiologia , Fator 2 Relacionado a NF-E2/metabolismo , Material Particulado/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Microscopia Confocal , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/metabolismoRESUMO
Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function.