RESUMO
BACKGROUND: Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI. METHODS: Long bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 µM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining. RESULTS: Our studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI. CONCLUSIONS: Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.
Assuntos
Lesões Encefálicas Traumáticas , Células Dendríticas , Cloreto de Amônio/metabolismo , Animais , Lesões Encefálicas Traumáticas/metabolismo , Diferenciação Celular , Desoxirribonuclease I/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Nylons/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: We found through previous research that hyperammonemia can cause secondary liver damage. However, whether hepatocytes are target cells of ammonia toxicity and whether hyperammonemia affects hepatocyte metabolism remain unknown. AIMS: The purpose of the current study is to examine whether the hepatocyte is a specific target cell of ammonia toxicity and whether hyperammonemia can interfere with hepatocyte metabolism. METHODS: Cell viability and apoptosis were analyzed in primary hepatocytes and other cells that had been exposed to ammonium chloride. Western blotting was adopted to examine the expression of proteins related to ammonia transport. We also established a metabolomics method based on gas chromatography-mass spectrometry to understand the characteristics of the hepatocyte metabolic spectrum in a hyperammonemia microenvironment, to screen and identify differential metabolites, and to determine the differential metabolic pathway. Different technologies were used to verify the differential metabolic pathways. RESULTS: Hepatocytes are target cells of ammonia toxicity. The mechanism is related to the ammonia transporter. Hyperammonemia interferes with hepatocyte metabolism, which leads to TCA cycle, urea cycle, and RNA synthesis disorder. CONCLUSIONS: This study demonstrates that hepatocyte growth and metabolism are disturbed in a hyperammonemia microenvironment, which further deteriorates hepatocyte function.
Assuntos
Hepatócitos/metabolismo , Hiperamonemia/metabolismo , Cloreto de Amônio/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular , Microambiente Celular , Ciclo do Ácido Cítrico , Cromatografia Gasosa-Espectrometria de Massas , Hepatócitos/citologia , Humanos , MetabolômicaRESUMO
The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.
Assuntos
Cloreto de Amônio/metabolismo , Glucose/metabolismo , Schizosaccharomyces/metabolismo , Corpos Polares do Fuso/metabolismo , Trifosfato de Adenosina/metabolismo , Centrômero/metabolismo , Schizosaccharomyces/citologiaRESUMO
SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH4Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH4Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH4Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiporters/genética , Membrana Celular/metabolismo , Distrofias Hereditárias da Córnea/genética , Mutação Puntual , Transporte Proteico/fisiologia , Cloreto de Amônio/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Linhagem Celular , Distrofias Hereditárias da Córnea/metabolismo , Cricetinae , Fibroblastos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , TransfecçãoRESUMO
This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.
Assuntos
Ácido Acético/metabolismo , Acil Coenzima A/metabolismo , Cloreto de Amônio/metabolismo , Ácido Glutâmico/metabolismo , Glioxilatos/metabolismo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/metabolismo , Carbono/metabolismo , Carboxiliases/metabolismo , Transporte de Elétrons/genética , Transporte de Elétrons/fisiologia , Perfilação da Expressão Gênica , Hidrogênio/metabolismo , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Nitrogênio/metabolismo , Rhodobacter capsulatus/crescimento & desenvolvimentoRESUMO
Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.
Assuntos
Cloreto de Amônio/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Acetil-CoA Carboxilase/genética , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Meios de Cultura/química , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Mitose/genética , Mutação , Penetrância , Fenótipo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels.
Assuntos
Amônia/química , Amônia/metabolismo , Aquaporina 4/química , Aquaporina 4/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Animais , Aquaporina 4/genética , Aquaporinas/química , Aquaporinas/genética , Aquaporinas/metabolismo , Canais Iônicos/genética , Transporte de Íons/fisiologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Oócitos , Ratos , Xenopus laevisRESUMO
BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. RESULTS: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. CONCLUSIONS: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15N-labelled amino acids could be obtained.
Assuntos
Aminoácidos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Aminoácidos/análise , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Meios de Cultura/química , Cromatografia Gasosa-Espectrometria de Massas , Marcação por Isótopo , Isótopos de Nitrogênio/químicaRESUMO
Eutrophication promotes massive growth of cyanobacteria and algal blooms, which can poison other algae and reduce biodiversity. To investigate the differences in multiple nitrogen (N) sources in eutrophicated water on the emission of volatile organic compounds (VOCs) from cyanobacteria, and their toxic effects on other algal growth, we analyzed VOCs emitted from Microcystis flos-aquae with different types and concentrations of nitrogen, and determined the effects under Normal-N and Non-N conditions on Chlorella vulgaris. M. flos-aquae released 27, 22, 20, 27, 19, 25 and 17 compounds, respectively, with NaNO3, NaNO2, NH4Cl, urea, Ser, Lys and Arg as the sole N source. With the reduction in N amount, the emission of VOCs was increased markedly, and the most VOCs were found under Non-N condition. C. vulgaris cell propagation, photosynthetic pigment and Fv/Fm declined significantly following exposure to M. flos-aquae VOCs under Non-N condition, but not under Normal-N condition. When C. vulgaris cells were treated with two terpenoids, eucalyptol and limonene, the inhibitory effects were enhanced with increasing concentrations. Therefore, multiple N sources in eutrophicated water induce different VOC emissions from cyanobacteria, and reduction in N can cause nutrient competition, which can result in emissions of more VOCs. Those VOCs released from M. flos-aquae cells under Non-N for nutrient competition can inhibit other algal growth. Among those VOCs, eucalyptol and limonene are the major toxic agents.
Assuntos
Chlorella vulgaris/efeitos dos fármacos , Chlorella vulgaris/fisiologia , Eutrofização , Microcystis/metabolismo , Nitrogênio/metabolismo , Compostos Orgânicos Voláteis/toxicidade , Cloreto de Amônio/metabolismo , Arginina/metabolismo , Chlorella vulgaris/crescimento & desenvolvimento , Cicloexanóis/farmacologia , Cicloexenos/farmacologia , Eucaliptol , Limoneno , Lisina/metabolismo , Monoterpenos/farmacologia , Nitratos/metabolismo , Fotossíntese/efeitos dos fármacos , Serina/metabolismo , Nitrito de Sódio/metabolismo , Terpenos/farmacologia , Ureia/metabolismo , Compostos Orgânicos Voláteis/análiseRESUMO
BACKGROUND: Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. RESULTS: The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. CONCLUSION: To our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.
Assuntos
Biomassa , Fermentação , Lipídeos/biossíntese , Yarrowia/metabolismo , Cloreto de Amônio/metabolismo , Reatores Biológicos , Glicerol/metabolismo , Ensaios de Triagem em Larga Escala , Metabolismo dos Lipídeos , Fatores de Tempo , Yarrowia/crescimento & desenvolvimentoRESUMO
As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.
Assuntos
Chlorella/crescimento & desenvolvimento , Chlorella/metabolismo , Modelos Biológicos , Nitrogênio/metabolismo , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Processos Autotróficos , Glutamato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Processos Heterotróficos , Cinética , Redes e Vias Metabólicas , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismoRESUMO
Trypanosoma cruzi, like other trypanosomatids analyzed so far, can use both glucose and amino acids as carbon and energy source. In these parasites, glycolysis is compartmentalized in glycosomes, authentic but specialized peroxisomes. The major part of this pathway, as well as a two-branched glycolytic auxiliary system, are present in these organelles. The first enzyme of one branch of this auxiliary system is the PPi-dependent pyruvate phosphate dikinase (PPDK) that converts phosphoenolpyruvate (PEP), inorganic pyrophosphate (PPi) and AMP into pyruvate, inorganic phosphate (Pi) and ATP, thus contributing to the ATP/ADP balance within the glycosomes. In this work we cloned, expressed and purified the T. cruzi PPDK. It kinetic parameters were determined, finding KM values for PEP, PPi and AMP of 320, 70 and 17 µM, respectively. Using molecular exclusion chromatography, two native forms of the enzyme were found with estimated molecular weights of 200 and 100 kDa, corresponding to a homodimer and monomer, respectively. It was established that T. cruzi PPDK's specific activity can be enhanced up to 2.6 times by the presence of ammonium in the assay mixture. During growth of epimastigotes in batch culture an apparent decrease in the specific activity of PPDK was observed. However, when its activity is normalized for the presence of ammonium in the medium, no significant modification of the enzyme activity per cell in time was found.
Assuntos
Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/enzimologia , Cloreto de Amônio/metabolismo , Animais , Doença de Chagas/parasitologia , Clonagem Molecular , Escherichia coli , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Microcorpos/metabolismo , Peso Molecular , Cloreto de Potássio/metabolismo , Piruvato Ortofosfato Diquinase/química , Piruvato Ortofosfato Diquinase/genética , Piruvato Ortofosfato Diquinase/isolamento & purificação , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/metabolismo , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: 2-phenylethanl (2-PE) and its derivatives are important chemicals, which are widely used in food materials and fine chemical industries and polymers and it's also a potentially valuable alcohol for next-generation biofuel. However, the biosynthesis of 2-PE are mainly biotransformed from phenylalanine, the price of which barred the production. Therefore, it is necessary to seek more sustainable technologies for 2-PE production. RESULTS: A new strain which produces 2-PE through the phenylpyruvate pathway was isolated and identified as Enterobacter sp. CGMCC 5087. The strain is able to use renewable monosaccharide as the carbon source and NH4Cl as the nitrogen source to produce 2-PE. Two genes of rate-limiting enzymes, chorismate mutase p-prephenate dehydratase (PheA) and 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (DAHP), were cloned from Escherichia coli and overexpressed in E. sp. CGMCC 5087. The engineered E. sp. CGMCC 5087 produces 334.9 mg L⻹ 2-PE in 12 h, which is 3.26 times as high as the wild strain. CONCLUSIONS: The phenylpyruvate pathway and the substrate specificity of 2-keto-acid decarboxylase towards phenylpyruvate were found in E. sp. CGMCC 5087. Combined with the low-cost monosaccharide as the substrate, the finding provides a novel and potential way for 2-PE production.
Assuntos
Enterobacter/metabolismo , Álcool Feniletílico/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Cloreto de Amônio/metabolismo , Cromatografia Gasosa , Escherichia coli/enzimologia , Monossacarídeos/metabolismo , Álcool Feniletílico/análise , Prefenato Desidrogenase/genética , Prefenato Desidrogenase/metabolismo , Especificidade por SubstratoRESUMO
A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.
Assuntos
Plásticos Biodegradáveis/metabolismo , Polietileno/química , Polietileno/efeitos da radiação , Poli-Hidroxialcanoatos/metabolismo , Cloreto de Amônio/metabolismo , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Plásticos Biodegradáveis/química , Temperatura Alta , Nitratos/metabolismo , Polietileno/metabolismo , Poli-Hidroxialcanoatos/químicaRESUMO
Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.
Assuntos
Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Celulase/metabolismo , Engenharia Metabólica/métodos , Mutagênese , Cloreto de Amônio/metabolismo , Aspergillus oryzae/metabolismo , Carbono/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , Oryza/metabolismo , Caules de Planta/metabolismo , Temperatura , Raios UltravioletaRESUMO
The reproductive toxicity potential of Ammonium Salt of Perfluorinated Hexanoic Acid (PFHxA Ammonium Salt) in pregnant Crl: CD1(ICR) mice was investigated. Twenty females/group were administered the test substance or vehicle once daily from gestation day 6 through 18. Phase 1 doses: 0, 100, 350, and 500 mg/kg/d; phase 2: 0, 7, 35, and 175 mg/kg/d. Parameters evaluated include mortality, viability, body weights, clinical signs, abortions, premature deliveries, pregnancy and fertility, litter observations, maternal behavior, and sexual maturity in the F1 generation. The level of PFHxA Ammonium Salt was measured in the liver of F0 and F1 mice. At doses of 350 and 500 mg/kg/d maternal mortalities, excess salivation and changes in body weight gains occurred. Pup body weights were reduced on postpartum day (PPD) 0 in all the dosage groups, but persisted only in the 350 and 500 mg/kg/d groups. Additional effects at 300 and 500 mg/kg/d included stillbirths, reductions in viability indices, and delays in physical development. Levels of PFHxA Ammonium Salt in the livers of the 100 mg/kg/d dams were all below the lower limit of quantization (0.02 µg/mL); in the 350 mg/kg/d group, 3 of the 8 samples had quantifiable analytical results. In phase 2 no PFHxA Ammonium Salt was found in the liver. Adverse effects occurred only in the 175 mg/kg/d group and consisted of increased stillborn pups, pups dying on PPD 1, and reduced pup weights on PPD 1. Based on these data, the maternal and reproductive no observable adverse effect level of PFHxA Ammonium Salt is 100 mg/kg/d.
Assuntos
Caproatos/toxicidade , Poluentes Ambientais/toxicidade , Desenvolvimento Fetal/efeitos dos fármacos , Fluorocarbonos/toxicidade , Infertilidade Feminina/induzido quimicamente , Infertilidade Masculina/induzido quimicamente , Exposição Materna/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal , Administração Oral , Cloreto de Amônio/administração & dosagem , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Cloreto de Amônio/toxicidade , Animais , Caproatos/administração & dosagem , Caproatos/química , Caproatos/metabolismo , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/química , Poluentes Ambientais/metabolismo , Feminino , Fluorocarbonos/administração & dosagem , Fluorocarbonos/química , Fluorocarbonos/metabolismo , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Lactação , Masculino , Camundongos , Nível de Efeito Adverso não Observado , Gravidez , Complicações na Gravidez/induzido quimicamente , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Maturidade Sexual/efeitos dos fármacos , Distribuição Tecidual , Toxicocinética , Aumento de Peso/efeitos dos fármacosRESUMO
As one of the most commonly used biocidal cationic surfactants, benzalkonium chlorides (BACs) have been an increasing concern as emerging contaminants. Wastewater has been claimed the main point for BACs to enter into the environment, but to date, it is still largely unknown how the BACs affect the microbes (especially microalgae) in the practical wastewater and how to cost-effectively remove them. In this study, the inhibitory effects of a typical BACs, dodecyl dimethyl benzyl ammonium chloride (DDBAC), on a green microalga Chlorella sp. in oxidation pond wastewater were investigated. The results showed that though a hermetic effect at the first 2 days was observed with the DDBAC at low concentration (<6 mg/L), the algal growth and photosynthesis were significantly inhibited by the DDBAC at all the tested concentrations (3 to 48 mg/L). Fortunately, a new microbial consortium (MC) capable of degrading DDBAC was screened through a gradient domestication method. The MC mainly composed of Wickerhamomyces sp., Purpureocillium sp., and Achromobacter sp., and its maximum removal efficiency and removal rate of DDBAC (48 mg/L) respectively reached 98.1 % and 46.32 mg/L/d. Interestingly, a microbial-microalgal system (MMS) was constructed using the MC and Chlorella sp., and a synergetic effect between the two kinds of microorganisms was proposed: microalga provided oxygen and extracellular polysaccharides as co-metabolic substrates to help the MC to degrade DDBAC, while the MC helped to eliminate the DDBAC-induced inhibition on the alga. Further, by observing the seven kinds of degradation products (mainly including CH5O3P, C6H5CH2-, and C8H11N), two possible chemical pathways of the DDBAC degradation were proposed. In addition, the metagenomic sequencing results showed that the main functional genes of the MMS included antibiotic-resistant genes, ABC transporter genes, quorum sensing genes, two-component regulatory system genes, etc. This study provided some theoretical and application findings for the cost-effective pollution prevention of BACs in wastewater.
Assuntos
Chlorella , Microalgas , Águas Residuárias , Cloreto de Amônio/metabolismo , Consórcios Microbianos , Chlorella/metabolismo , Técnicas de Cocultura , BiomassaRESUMO
Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 µM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.
Assuntos
Amilorida/análogos & derivados , Proteínas de Transporte de Ânions/antagonistas & inibidores , Antiporters/antagonistas & inibidores , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Sódio/metabolismo , Amilorida/farmacologia , Sequência de Aminoácidos , Cloreto de Amônio/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antiporters/genética , Antiporters/metabolismo , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Dados de Sequência Molecular , Hidróxido de Sódio/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked ß-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Glutamato Desidrogenase/isolamento & purificação , Mucorales/enzimologia , Cloreto de Amônio/metabolismo , Antifúngicos/síntese química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/ultraestrutura , Catálise , Cromatografia em Agarose , Avaliação Pré-Clínica de Medicamentos , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Histidina/química , Histidina/efeitos dos fármacos , Hifas/enzimologia , Ponto Isoelétrico , Ácidos Cetoglutáricos/metabolismo , Lisina/química , Lisina/efeitos dos fármacos , Terapia de Alvo Molecular , Peso Molecular , Mucorales/efeitos dos fármacos , Mucorales/fisiologia , Mucorales/ultraestrutura , NAD/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Triazóis/farmacologia , Yarrowia/efeitos dos fármacos , Yarrowia/enzimologia , Yarrowia/ultraestruturaRESUMO
In this study, Na(+) uptake and acid-base balance in the euryhaline pupfish Cyprinodon variegatus variegatus were characterized when fish were exposed to pH 4.5 freshwater (7mM Na(+)). Similar to the related cyprinodont, Fundulus heteroclitus, Na(+) uptake was significantly inhibited when exposed to low pH water. However, it initially appeared that C. v. variegatus increased apparent net acid excretion at low pH relative to circumneutral pH. This result is opposite to previous observations for F. heteroclitus under similar conditions where fish were observed to switch from apparent net H(+) excretion at circumneutral pH to apparent net H(+) uptake at low pH. Further investigation revealed disparate observations between these studies were the result of using double endpoint titrations to measure titratable alkalinity fluxes in the current study, while the earlier study utilized single endpoint titrations to measure these fluxes (i.e.,. Cyprinodon acid-base transport is qualitatively similar to Fundulus when characterized using single endpoint titrations). This led to a comparative investigation of these two methods. We hypothesized that either the single endpoint methodology was being influenced by a change in the buffer capacity of the water (e.g., mucus being released by the fish) at low pH, or the double endpoint methodology was not properly accounting for ammonia flux by the fish. A series of follow-up experiments indicated that buffer capacity of the water did not change significantly, that excretion of protein (a surrogate for mucus) was actually reduced at low pH, and that the double endpoint methodology does not properly account for NH(3) excretion by fish under low pH conditions. As a result, it overestimates net H(+) excretion during low pH exposure. After applying the maximum possible correction for this error (i.e., assuming that all ammonia is excreted as NH(3)), the double endpoint methodology indicates that net H(+) transport was reduced to effectively zero in both species at pH 4.5. However, significant differences between the double endpoint (no net H(+) transport at low pH) and single endpoint titrations (net H(+) uptake at low pH) remain to be explained.