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The ecotoxicological effects of herbicide contamination on the autotrophic growth of microalgae in aquatic environments have been major concerns. However, little is known about the influence of herbicides on the mixotrophic growth of microalgae. This study investigated the ecotoxicological effect of 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) on the mixotrophic growth of Chlorella sacchrarophila FACHB 4. Results showed that C. sacchrarophila in mixotrophy was more resistant to DCMU than in photoautotrophy. Moreover, a low content of DCMU (20-80 µg·L-1) promoted the mixotrophic growth of C. sacchrarophila, and the promotion effect was obviously enhanced with the increase in light intensity. The chlorophyll content and glucose absorption rate of C. sacchrarophila were found to increase after incubation with DCMU for 24 h. Transcriptome analyses revealed that the mechanism of DCMU to promote the mixotrophic growth of C. sacchrarophila was probably through accelerating glucose uptake and utilization, which was accomplished by reducing photodamage and increasing the chlorophyll content of C. sacchrarophila. This study not only revealed an unexpected bloom of mixotrophic microalgae triggered by herbicides, but it also shed new light on an effective and low-cost strategy to improve the microalgae productivity for utilization.
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Chlorella , Herbicidas , Microalgas , Herbicidas/toxicidad , Diurona , Biomasa , ClorofilaRESUMEN
Recent investigations demonstrate that some coastal wetlands are atmospheric methane sinks, but the regulatory mechanisms are not clear. Here, the main pathway and operator of methane oxidation in the Yellow River Delta (YRD) wetland, a methane source in the wet season but a methane sink in the dry season, were investigated. The anaerobic oxidation of methane (AOM) and aerobic methane oxidation (AMO) abilities of wetland soil were measured, and the microbial community structure was analyzed. The experimental results showed that AMO was active throughout the year. In contrast, AOM was weak and even undetected. The microbial community analysis indicated that Methylomicrobium and Methylobacter potentially scavenged methane in oxic environments. A representative strain of Methylobacter, which was isolated from the soil, presented a strong AMO ability at high concentrations of methane and air. Overall, this study showed that active AMO performing by Methylobacter may account for methane sink in the YRD wetland during the dry season. Our research not only has determined the way in which methane sinks are formed but also identified the potential functional microbes. In particular, we confirmed the function of potential methanotroph by pure culture. Our research provides biological evidence for why some wetlands have methane sink characteristics, which may help to understand the global methane change mechanism.
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Metano/metabolismo , Methylococcaceae/metabolismo , Contaminantes Químicos del Agua/metabolismo , Aerobiosis , Biodegradación Ambiental , China , Ríos/química , Ríos/microbiología , Microbiología del AguaRESUMEN
Conductive materials/minerals can promote direct interspecies electron transfer (DIET) between syntrophic bacteria and methanogens in defined co-culture systems and artificial anaerobic digesters; however, little is known about the stimulation strategy of carbon material on methane production in natural environments. Herein, the effect of carbon cloth, as a representative of conductive carbon materials, on methane production with incubated wetland soil was investigated. Carbon cloth significantly promoted methanogenesis. With the application of electrochemical technology, calculation of the apparent electron transfer rate constant showed that carbon cloth significantly increased electron transfer rate (ETR) compared with the control experiment in presence of cotton cloth, from 0.0017⯱â¯0.0003 to 0.0056⯱â¯0.0015â¯s-1. Results obtained from both stable carbon isotope measurements and application of specific inhibitor (CH3F) for acetoclastic methanogenesis indicated that carbon cloth obviously promoted acetoclastic methanogenesis instead of CO2 reduction. High-throughput sequencing showed that methane production may stem from the involvement of Methanosarcina for both treatments. Our findings suggested that conductive carbon material can promote acetoclastic methanogenesis instead of CO2 reduction in a natural environment.
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Fascin2 (FSCN2) is an actin cross-linking protein that is mainly localized in retinas and in the stereocilia of hair cells. Earlier studies showed that a deletion mutation in human FASCIN2 (FSCN2) gene could cause autosomal dominant retinitis pigmentosa. Recent studies have indicated that a missense mutation in mouse Fscn2 gene (R109H) can contribute to the early onset of hearing loss in DBA/2J mice. To explore the function of the gene, Fscn2 was knocked out using TALEN (transcription activator-like effector nucleases) on the C57BL/6J background. Four mouse strains with deletions of 1, 4, 5, and 41 nucleotides in the target region of Fscn2 were developed. F1 heterozygous (Fscn2+/- ) mice carrying the same deletion of 41 nucleotides were mated to generate the Fscn2-/- mice. As a result, the Fscn2-/- mice showed progressive hearing loss, as measured in the elevation of auditory brainstem-response thresholds. The hearing impairment began at age 3 weeks at high-stimulus frequencies and became most severe at age 24 weeks. Moreover, degeneration of hair cells and loss of stereocilia were remarkable in Fscn2-/- mice, as revealed by F-actin staining and scanning electron microscopy. Furthermore, compared to the controls, the Fscn2-/- mice displayed significantly lower electroretinogram amplitudes and thinner retinas at 8, 16, and 24 weeks. These results demonstrate that, in C57BL/6Jmice, Fscn2 is essential for maintaining ear and eye function and that a null mutation of Fscn2 leads to progressive hearing loss and retinal degeneration.
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Proteínas Portadoras/genética , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Homocigoto , Proteínas de Microfilamentos/genética , Mutación , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Animales , Secuencia de Bases , Biopsia , Recuento de Células , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrorretinografía , Expresión Génica , Frecuencia de los Genes , Marcación de Gen , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/ultraestructura , Pérdida Auditiva/diagnóstico , Heterocigoto , Ratones , Ratones Endogámicos C57BL , Fenotipo , Degeneración Retiniana/diagnóstico , Eliminación de SecuenciaRESUMEN
BACKGROUND: Magnetite-mediated direct interspecies electron transfer (DIET) between Geobacter and Methanosarcina species is increasingly being invoked to explain magnetite stimulation of methane production in anaerobic soils and sediments. Although magnetite-mediated DIET has been documented in defined co-cultures reducing fumarate or nitrate as the electron acceptor, the effects of magnetite have only been inferred in methanogenic systems. METHODS: Concentrations of methane and organic acid were analysed with a gas chromatograph and high-performance liquid chromatography, respectively. The concentration of HCl-extractable Fe(II) was determined by the ferrozine method. The association of the defined co-cultures of G. metallireducens and M. barkeri with magnetite was observed with transmission electron micrographs. RESULTS: Magnetite stimulated ethanol metabolism and methane production in defined co-cultures of G. metallireducens and M. barkeri; however, magnetite did not promote methane production in co-cultures initiated with a culture of G. metallireducens that could not produce electrically conductive pili (e-pili), unlike the conductive carbon materials that facilitate DIET in the absence of e-pili. Transmission electron microscopy revealed that G. metallireducens and M. barkeri were closely associated when magnetite was present, as previously observed in G. metallireducens/G. sulfurreducens co-cultures. These results show that magnetite can promote DIET between Geobacter and Methanosarcina species, but not as a substitute for e-pili, and probably functions to facilitate electron transfer from the e-pili to Methanosarcina. CONCLUSION: In summary, the e-pili are necessary for the stimulation of not only G. metallireducens/G. sulfurreducens, but also methanogenic G. metallireducens/M. barkeri co-cultures with magnetite.
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The erl mouse is a mouse model of nonsyndromic autosomal recessive deafness (DFNB12) on the C57BL/6J background. This project was carried out to express the first two ectodomains of cadherin 23 (CDH23 EC1+2) of erl mice in Escherichia coli and identify the Ca2+-binding ability of the recombinant protein. DNA sequences of CDH23 EC1+2 from wild type and erl mice were synthesized and cloned into pBV220 plasmids. Recombinant plasmids were transformed into Escherichia coli and expression of CDH23 EC1+2 was induced by increasing the temperature from 30 °C to 42 °C. The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and antigenicity of proteins was identified by Western Blotting. Inclusion bodies were denatured in 8 M urea, purified by ion-exchange and gel filtration chromatography and refolded with dialysis in buffer containing 0.1% sarkosyl. The Ca2+-binding ability of CDH23 EC1+2 was determined by Ca2+-dependent proteolysis protection. The results showed that the sizes and sequences of inserts in recombinant plasmids were consistent with expectation and that the recombinant proteins were found mainly in the form of inclusion bodies which maintain antigenicity. After refolding, the secondary structures of recombinant proteins were measured by circular dichroism (CD) spectra. Moreover, CDH23 EC1+2 from the erl mice showed less Ca2+-dependent proteolysis protection comparing with that of the wild type control. We therefore concluded that impairment of Ca2+-dependent protein interaction was likely involved in the progressive hearing loss in erl mice. The results may aid in understanding the mechanism of hearing loss in DFNB12.
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Cadherinas/metabolismo , Calcio/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cadherinas/química , Cadherinas/genética , Pérdida Auditiva Sensorineural/genética , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Replegamiento Proteico , Estructura Secundaria de Proteína , Proteolisis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Both activated carbon and magnetite have been reported to promote the syntrophic growth of Geobacter metallireducens and Geobacter sulfurreducens co-cultures, the first model to show direct interspecies electron transfer (DIET); however, differential transcriptomics of the promotion on co-cultures with these two conductive materials are unknown. Here, the comparative transcriptomic analysis of G. metallireducens and G. sulfurreducens co-cultures with granular activated carbon (GAC) and magnetite was reported. More than 2.6-fold reduced transcript abundances were determined for the uptake hydrogenase genes of G. sulfurreducens as well as other hydrogenases in those co-cultures to which conductive materials had been added. This is consistent with electron transfer in G. metallireducens-G. sulfurreducens co-cultures as evinced by direct interspecies electron transfer (DIET). Transcript abundance for the structural component of electrically conductive pili (e-pili), PilA, was 2.2-fold higher in G. metallireducens, and, in contrast, was 14.9-fold lower in G. sulfurreducens in co-cultures with GAC than in Geobacters co-cultures without GAC. However, it was 9.3-fold higher in G. sulfurreducens in co-cultures with magnetite than in Geobacters co-cultures. Mutation results showed that GAC can be substituted for the e-pili of both strains but magnetite can only compensate for that of G. sulfurreducens, indicating that the e-pili is a more important electron acceptor for the electron donor strain of G. metallireducens than for G. sulfurreducens. Transcript abundance for G. metallireducens c-type cytochrome gene GMET_RS14535, a homologue to c-type cytochrome gene omcE of G. sulfurreducens was 9.8-fold lower in co-cultures with GAC addition, while that for OmcS of G. sulfurreducens was 25.1-fold higher in co-cultures with magnetite, than in that without magnetite. Gene deletion studies showed that neither GAC nor magnetite can completely substitute the cytochrome (OmcE homologous) of G. metallireducens but compensate for the cytochrome (OmcS) of G. sulfurreducens. Moreover, some genes associated with central metabolism were up-regulated in the presence of both GAC and magnetite; however, tricarboxylic acid cycle gene transcripts in G. sulfurreducens were not highly-expressed in each of these amended co-cultures, suggesting that there was considerable redundancy in the pathways utilised by G. sulfurreducens for electron transfer to reduce fumarate with the amendment of GAC or magnetite. These results support the DIET model of G. metallireducens and G. sulfurreducens and suggest that e-pili and cytochromes of the electron donor strain are more important than that of the electron acceptor strain, indicating that comparative transcriptomics may be a promising route by which to reveal different responses of electron donor and acceptor during DIET in co-cultures.
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Carbón Orgánico/metabolismo , Transporte de Electrón/genética , Óxido Ferrosoférrico/metabolismo , Geobacter/genética , Transcriptoma , Proteínas de la Membrana Bacteriana Externa/genética , Técnicas de Cocultivo , Fimbrias Bacterianas/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Geobacter/clasificación , Geobacter/crecimiento & desarrollo , Geobacter/metabolismo , Oxidación-ReducciónRESUMEN
Minerals that contain ferric iron, such as amorphous Fe(III) oxides (A), can inhibit methanogenesis by competitively accepting electrons. In contrast, ferric iron reduced products, such as magnetite (M), can function as electrical conductors to stimulate methanogenesis, however, the processes and effects of magnetite production and transformation in the methanogenic consortia are not yet known. Here we compare the effects on methanogenesis of amorphous Fe (III) oxides (A) and magnetite (M) with ethanol as the electron donor. RNA-based terminal restriction fragment length polymorphism with a clone library was used to analyse both bacterial and archaeal communities. Iron (III)-reducing bacteria including Geobacteraceae and methanogens such as Methanosarcina were enriched in iron oxide-supplemented enrichment cultures for two generations with ethanol as the electron donor. The enrichment cultures with A and non-Fe (N) dominated by the active bacteria belong to Veillonellaceae, and archaea belong to Methanoregulaceae and Methanobacteriaceae, Methanosarcinaceae (Methanosarcina mazei), respectively. While the enrichment cultures with M, dominated by the archaea belong to Methanosarcinaceae (Methanosarcina barkeri). The results also showed that methanogenesis was accelerated in the transferred cultures with ethanol as the electron donor during magnetite production from A reduction. Powder X-ray diffraction analysis indicated that magnetite was generated from microbial reduction of A and M was transformed into siderite and vivianite with ethanol as the electron donor. Our data showed the processes and effects of magnetite production and transformation in the methanogenic consortia, suggesting that significantly different effects of iron minerals on microbial methanogenesis in the iron-rich coastal riverine environment were present.
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Óxido Ferrosoférrico/metabolismo , Sedimentos Geológicos/microbiología , Metano/metabolismo , Methanomicrobiales/metabolismo , Consorcios Microbianos/fisiología , Ríos/microbiología , Anaerobiosis , Compuestos Férricos/metabolismo , Compuestos Férricos/farmacología , Geobacter/efectos de los fármacos , Geobacter/metabolismo , Hierro/metabolismo , Methanomicrobiales/efectos de los fármacos , Methanosarcina/efectos de los fármacos , Methanosarcina/metabolismo , ARN Ribosómico 16S/genéticaRESUMEN
Iron (III)-reducing bacteria (IRB) play significant roles in the degradation of naturally occurring organic matter and in the cycling of heavy metals in marine and freshwater sediments. Our previous study has demonstrated the co-occurrence of Geobacteraceae and Methanosarcinamazei as aggregates in the iron (III)-reducing enrichments from a coastal gold mining site on the Jiehe River. The IRB community in the enriched sediments was dominated by members of Comamonadacea, Clostridiaceae, Bacillaceae, Bacteroidaceae, and Geobacteraceae. Furthermore, four representative strains (JhA, JhB, JhC-1, and JhC-2) were isolated and found to belong to the genus of Anaerosinus, Bacillus, and Clostridium with 97.31-98.82% identity of 16S rRNA genes. The iron (III)-reducing ability of all these isolates was identified. Interestingly, JhA showed electrochemical activity with chronoamperometry (CA) and cyclic voltammetry (CV), indicating its ability to oxidize ethanol, liberate, and transfer electrons, thus, expanding our knowledge of a new genus with electrochemical activity. The results revealed the cultivability and electrochemical activity of IRB in coastal riverine sediment and indicated that JhA was an unknown extracellular electron producer with the ability to reduce iron (III). This study expands our knowledge of the electrochemical characterization of the genus Anaerosinus. It is reasonable to expect that these isolates have potential applications in heavy metal bioremediation operations in natural environments.
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Bacterias/clasificación , Bacterias/metabolismo , Biota , Sedimentos Geológicos/microbiología , Hierro/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Atmospheric nitrogen deposition caused by human activities has been receiving much attention. Here, after long-term simulated ammonium and nitrate nitrogen deposition (NH4Cl, KNO3, and NH4NO3) in the Yellow River Delta (YRD), a sensitive coastal wetland ecosystem typified by a distinct wet and dry season, methane fluxes were measured, by adopting a closed static chamber technique. The results showed that deposition of ammonium nitrogen accelerated methane emissions all year round. Ammonium nitrogen deposition transformed the YRD from a methane sink into a source during the dry season. Methanocellaceae is the only methanogen with increased abundance after the application of NH4Cl and NH4NO3, which promoted methane emissions, during the wet season. The findings suggested that Methanocellaceae may facilitate methane emissions in response to increased ammonium nitrogen deposition. Other methanogens might have profited from ammonium supplementation, such as Methanosarcinaceae. Deposition of nitrate nitrogen did not affect methane flux significantly. To the best of our knowledge, this study is the first to show that Methanocellaceae may be responsible for methane production in coastal wetland system. This study highlights the significant effect of ammonium nitrogen and slight effect of nitrate nitrogen on methane emission in the YRD and it will be helpful to understand the microbial mechanism responding to increased nitrogen deposition in the sensitive coastal wetland ecosystem.
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Compuestos de Amonio/metabolismo , Metano/biosíntesis , Methanomicrobiaceae/metabolismo , Nitrógeno/metabolismo , Humedales , China , RíosRESUMEN
Wetland-estuarine-marine environments are typical oxic/anoxic transition zones and have complex water flow-paths within the zone of mixing where freshwater interacts with ocean water. Little is known about the impact of this interaction on bacterial community structures or the relationship between bacterial community and geochemical factors in such transitional mixing environments. Hence, we investigated the distribution patterns and diversity in bacterial communities in the Yellow River estuary-coastal wetland-Bohai Sea transition zone by analyzing 39 samples from 13 ordered sites. High-throughput sequencing of the 16S rRNA gene revealed significant shifts in diversity and distribution of bacterial community in sediments from the Yellow River estuary to the Bohai Sea. Yellow River sediment was dominated by hydrogen-, nitrogen-, and iron-cycling bacteria, such as Hydrogenophaga, Nitrospira, Pseudomonas, and Thiobacillus. The coastal wetland had a haloduric community associated with different functions, such as Planctomyces, Marinobacter, Halomonas, Salinivibrio, and Salinibacter. The Bohai Sea sediment had a higher relative abundance of Lutimonas, Desulfococcus, Photobacterium, Propionigenium, and Vibrio. Spatial variation in bacterial community was correlated with pH, salinity and sulfate (SO42-) concentration in such coastal environments. The major bacterial taxa were significantly different across the wetland, estuary, and coastal marine ecosystems, indicating substantial spatial heterogeneity among the three ecosystems. Statistical analysis revealed strong links between variation in bacterial community structure and ecosystem type. Our results demonstrate the importance of geographic and geochemical factors in structuring the bacterial community in natural environments.
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Biodiversidad , Ecosistema , Consorcios Microbianos , Microbiología del Agua , Humedales , Bacterias/genética , Bacterias/aislamiento & purificación , ADN Bacteriano , Estuarios , Sedimentos Geológicos/microbiología , Filogenia , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Ríos/microbiología , Salinidad , Agua de Mar/microbiología , Análisis de Secuencia de ADN , Thiobacillus/genética , Thiobacillus/aislamiento & purificaciónRESUMEN
OBJECTIVES: To investigate the otoprotective effects of mouse nerve growth factor (mNGF) in A/J mice. METHODS: The mice at postnatal day 7 (P7) were randomly separated into a mNGF treated group (mNGF group) and a distilled water (for injection) treated group (control group). The mNGF dissolved in distilled water or distilled water alone was given to the mice once every other day from P7 by intramuscular injection in the hips. The otoprotective effects of mNGF in A/J mice were observed in a time course manner. The thresholds of auditory-evoked brainstem response (ABR) were tested from the age of the 3rd to the 8th week. Sections of the inner ears were stained by hematoxylin and eosin, and spiral ganglion neurons (SGNs) were observed at the age of the 3rd, the 6th,and the 8th week. Counts of whole mount outer hair cells (OHCs) in the cochleae were made at the age of 8 weeks. Expression of apoptosis related genes was determined by quantitative real-time polymerase chain reaction and Western blotting. RESULTS: ABR thresholds of the mNGF group were significantly lower than those of the control group at the age of the 6th and the 8th week. Moreover, the mNGF preserved OHC and SGN in the mouse cochleae in this period. Further experiments showed that the expression of caspase genes (including caspase-3) was inhibited in the mouse inner ears in the mNGF group. CONCLUSION: The mNGF improves hearing in A/J mice by preserving SGN and OHC in the cochleae.
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Age-related hearing loss (AHL), or presbycusis, is the most common neurodegenerative disorder and top communication deficit of the aged population. Genetic predisposition is one of the major factors in the development of AHL. Generally, AHL is associated with an age-dependent loss of sensory hair cells, spiral ganglion neurons and stria vascularis cells in the inner ear. Although the mechanisms leading to genetic hearing loss are not completely understood, caspase-family proteases function as important signals in the inner ear pathology. It is now accepted that mouse models are the best tools to study the mechanism of genetic hearing loss or AHL. Here, we provide a brief review of recent studies on hearing improvement in mouse models of AHL by anti-apoptotic treatment.
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Methanosaeta harundinacea and Methanosarcina barkeri, known as classic acetoclastic methanogens, are capable of directly accepting electrons from Geobacter metallireducens for the reduction of carbon dioxide to methane, having been revealed as direct interspecies electron transfer (DIET) in the laboratory co-cultures. However, whether their co-occurrences are ubiquitous in the iron (III)-reducing environments and the other species of acetoclastic methanogens such as Methanosarcina mazei are capable of DIET are still unknown. Instead of initiating the co-cultures with pure cultures, two-step cultivation was employed to selectively enrich iron (III)-reducing microorganisms in a coastal gold mining river, Jiehe River, with rich iron content in the sediments. First, iron (III) reducers including Geobacteraceae were successfully enriched by 3-months successive culture on amorphous Fe(III) oxides as electron acceptor and acetate as electron donor. High-throughput Illumina sequencing, terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures actively contained the bacteria belong to Geobacteraceae and Bacilli, exclusively dominated by the archaea belong to Methanosarcinaceae. Second, the enrichment cultures including methanogens and Geobacteraceae were transferred with ethanol as alternative electron donor. Remarkably, aggregates were successively formed in the enrichments after three transfers. The results revealed by RNA-based analysis demonstrate that the co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture. Furthermore, the aggregates, as close physical contact, formed in the enrichment culture, indicate that DIET could be a possible option for interspecies electron transfer in the aggregates.
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Pancreatic derived factor (PANDER, FAM3B) is a peptide mainly synthesized and secreted by pancreatic ß-cells. PANDER is proposed to be involved in regulation of ß-cell function under physiological conditions and impairment of ß-cell function under pathological conditions. MCP-1 (CCL2) is expressed by normal pancreatic islets and has been implicated in inflammation related pancreatic disorders. We examined the effect of MCP-1 on PANDER expression by using murine pancreatic ß-cell line MIN6 and pancreatic islets. We found that MCP-1 induced PANDER mRNA transcription and protein synthesis in MIN6 cells and islets. By using calcium chelator (EGTA); inhibitors for PKC (Go6976), MEK1/2 (PD98059) or c-Jun-N-terminal kinase (JNK) (SP600125); c-Jun dominant-negative construct; PANDER promoter luciferase constructs; and islets isolated from Fos knockout mice; we demonstrated that MCP-1 induced PANDER gene expression in ß-cells through Ca(2+)-ERK1/2-AP-1 and PKC-JNK-AP-1 signaling pathways. Our findings suggest a new link between the endocrine and immune systems and provide useful information for further investigating the physiological functions of PANDER and its involvement in inflammation-related pancreatic disorders.
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Quimiocina CCL2/farmacología , Citocinas/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Calcio/metabolismo , Línea Celular , Citocinas/genética , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/citología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Amylin is the most abundant component of islet amyloid implicated in the development of type 2 diabetes. Plasma amylin levels are elevated in individuals with obesity and insulin resistance. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is involved in insulin resistance of obesity and type 2 diabetes. We investigated the effect of MCP-1 on amylin expression and the underlying mechanisms with murine pancreatic ß-cell line MIN6 and pancreatic islets. METHODOLOGY/PRINCIPAL FINDINGS: We found that MCP-1 induced amylin expression at transcriptional level and increased proamylin and intermediate forms of amylin at protein level in MIN6 cells and islets. However, MCP-1 had no effect on the expressions of proinsulin 1 and 2, as well as prohormone convertase (PC) 1/3 and PC2, suggesting that MCP-1 specifically induces amylin expression in ß-cells. Mechanistic studies showed that although there is no detectable CCR2 mRNA in MIN6 cells and islets, pretreatment of MIN6 cells with pertussis toxin inhibited MCP-1 induced amylin expression, suggesting that alternative Gi-coupled receptor(s) mediates the inductive effect of MCP-1. MCP-1 rapidly induced ERK1/2 and JNK phosphorylation. Inhibitors for MEK1/2 (PD98059), JNK (SP600125) or AP1 (curcumin) significantly inhibited MCP-1-induced amylin mRNA expression. MCP-1 failed to induce amylin expression in pancreatic islets isolated from Fos knockout mice. EMSA showed that JNK and ERK1/2 were involved in MCP-1-induced AP1 activation. These results suggest that MCP-1 induces murine amylin expression through AP1 activation mediated by ERK1/2 or JNK. Further studies showed that treatment of MIN6 cells with NF-κB inhibitor or overexpression of IκBα dominant-negative construct in MIN6 cells significantly inhibited MCP-1-induced amylin expression, suggesting that NF-κB related signaling also participates in MCP-1-induced murine amylin expression. CONCLUSIONS/SIGNIFICANCE: MCP-1 induces amylin expression through ERK1/2/JNK-AP1 and NF-κB related signaling pathways independent of CCR2. Amylin upregulation by MCP-1 may contribute to elevation of plasma amylin in obesity and insulin resistance.
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Quimiocina CCL2/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/metabolismo , Receptores CCR2/metabolismo , Transducción de Señal , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Islotes Pancreáticos/enzimología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Regulación hacia ArribaRESUMEN
Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic beta-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by beta-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic beta-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca(2+) chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca(2+) and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.
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Amiloide , Ácidos Grasos no Esterificados/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Transducción de Señal/fisiología , Amiloide/genética , Amiloide/metabolismo , Animales , Glucemia/metabolismo , Calcio/metabolismo , Células Cultivadas , Ácidos Grasos no Esterificados/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Polipéptido Amiloide de los Islotes Pancreáticos , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Oléico/metabolismo , Ácido Oléico/farmacología , Palmitatos/metabolismo , Palmitatos/farmacología , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.
Asunto(s)
Antioxidantes/farmacología , Catequina/análogos & derivados , Monocitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Camellia sinensis/inmunología , Catequina/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Flavonoides/farmacología , Humanos , Inflamación , Inyecciones Intraperitoneales , Leucemia Basofílica Aguda/sangre , Leucemia Basofílica Aguda/tratamiento farmacológico , Leucemia Basofílica Aguda/inmunología , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , N-Formilmetionina Leucil-Fenilalanina/inmunología , Ratas , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/inmunología , Receptores de Formil Péptido/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Transfección , TransgenesRESUMEN
Pancreatic-derived factor (PANDER) is a cytokine-like peptide highly expressed in pancreatic beta-cells. PANDER was reported to promote apoptosis of pancreatic beta-cells and secrete in response to glucose. Here we explored the effects of glucose on PANDER expression, and the underlying mechanisms in murine pancreatic beta-cell line MIN6 and primary islets. Our results showed that glucose up-regulated PANDER mRNA and protein levels in a time- and dose-dependent manner in MIN6 cells and pancreatic islets. In cells expressing cAMP response element-binding protein (CREB) dominant-negative construct, glucose failed to induce PANDER gene expression and promoter activation. Treatment of the cells with calcium chelator [EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM)], the voltage-dependent Ca(2+) channel inhibitor (nifedipine), the protein kinase A (PKA) inhibitor (H89), the protein kinase C (PKC) inhibitor (Go6976), or the MAPK kinase 1/2 inhibitor (PD98059), all significantly inhibited glucose-induced PANDER gene expression and promoter activation. Further studies showed that glucose induced CREB phosphorylation through Ca(2+)-PKA-ERK1/2 and Ca(2+)-PKC pathways. Thus, the Ca(2+)-PKA-ERK1/2-CREB and Ca(2+)-PKC-CREB signaling pathways are involved in glucose-induced PANDER gene expression. Wortmannin (phosphatidylinositol 3-kinase inhibitor), ammonium pyrrolidinedithiocarbamate (nuclear factor-kappaB inhibitor and nonspecific antioxidant), and N-acetylcysteine (antioxidant) were also found to inhibit glucose-induced PANDER promoter activation and gene expression. Because there is no nuclear factor-kappaB binding site in the promoter region of PANDER gene, these results suggest that phosphatidylinositol 3-kinase and reactive oxygen species be involved in glucose-induced PANDER gene expression. In conclusion, glucose induces PANDER gene expression in pancreatic beta-cells through multiple signaling pathways. Because PANDER is expressed by pancreatic beta-cells and in response to glucose in a similar way to those of insulin, PANDER may be involved in glucose homeostasis.
Asunto(s)
Citocinas/genética , Citocinas/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Glucosa/farmacología , Células Secretoras de Insulina/citología , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , TransfecciónRESUMEN
Transforming growth factor-beta (TGF-beta) family members are multifunctional molecules, which play pivotal roles in regulating cell proliferation, differentiation, migration, development, tissue remodeling and repair. These events are closely associated with host immune responses and inflammation. Despite some controversies on their function in controlling dendritic and T regulatory cell development and activity, the importance of TGF-betas in the progress of autoimmunity and inflammatory diseases has been well appreciated and new aspects of their contribution continue to be recognized. Since one of the major biological properties of TGF-betas is its capacity to potently suppress immune responses, they are considered as candidates for the development of therapeutic agents to fend off undesirable damage associated with immune and inflammatory conditions.