RESUMEN
Decarbonatization initiatives have rapidly increased the demand for lithium. This study uses public waste compliance reports and Monte Carlo approaches to estimate total lithium mass yields from produced water (PW) sourced from the Marcellus Shale in Pennsylvania (PA). Statewide, Marcellus Shale PW has substantial extractable lithium, however, concentrations, production volumes and extraction efficiencies vary between the northeast and southwest operating zones. Annual estimates suggest statewide lithium mass yields of approximately 1160 (95% CI 1140-1180) metric tons (mt) per year. Production decline curve analysis on PW volumes reveal cumulative volumetric disparities between the northeast (median = 2.89 X 107 L/10-year) and southwest (median = 5.56 × 107 L/10-year) regions of the state, influencing lithium yield estimates of individual wells in southwest [2.90 (95% CI 2.80-2.99) mt/10-year] and northeast [1.96 (CI 1.86-2.07) mt/10-year] PA. Moreover, Mg/Li mass ratios vary regionally, where NE PA are low Mg/Li fluids, having a median Mg/Li mass ratio of 5.39 (IQR, 2.66-7.26) and SW PA PW is higher with a median Mg/Li mass ratio of 17.8 (IQR, 14.3-20.7). These estimates indicate substantial lithium yields from Marcellus PW, though regional variability in chemistry and production may impact recovery efficiencies.
RESUMEN
Although zinc deficiency (secondary to malnutrition) has long been considered an important contributor to morbidity and mortality of infectious disease (e.g. diarrhea disorders), epidemiologic data (including randomized controlled trials with supplemental zinc) for such a role in lower respiratory tract infection are somewhat ambiguous. In the current study, we provide the first preclinical evidence demonstrating that although diet-induced acute zinc deficiency (Zn-D: ~50% decrease) did not worsen infection induced by either influenza A (H1N1) or methicillin-resistant staph aureus (MRSA), Zn-D mice were sensitive to the injurious effects of superinfection of H1N1 with MRSA. Although the mechanism underlying the sensitivity of ZnD mice to combined H1N1/MRSA infection is unclear, it was noteworthy that this combination exacerbated lung injury as shown by lung epithelial injury markers (increased BAL protein) and decreased genes related to epithelial integrity in Zn-D mice (surfactant protein C and secretoglobins family 1A member 1). As bacterial pneumonia accounts for 25%-50% of morbidity and mortality from influenza A infection, zinc deficiency may be an important pathology component of respiratory tract infections.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Desnutrición , Staphylococcus aureus Resistente a Meticilina , Neumonía Bacteriana , Animales , Ratones , Neumonía Bacteriana/complicaciones , Staphylococcus aureus , ZincRESUMEN
Nearly 30% of adults consume less than the estimated average daily requirement of magnesium (Mg2+), and commonly used medications, such as diuretics, promote Mg2+ deficiency. Higher serum Mg2+ levels, increased dietary Mg2+ in-take, and Mg2+ supplementation are each associated with lower blood pressure, suggesting that Mg2+-deficiency contributes to the pathogenesis of hypertension. Antigen-presenting cells, such as monocytes and dendritic cells, are well-known to be involved in the pathogenesis of hypertension. In these cells, processes implicated as necessary for increased blood pressure include activation of the NLRP3 inflammasome, IL-1ß production, and oxidative modification of fatty acids such as arachidonic acid, forming isolevuglandins (IsoLGs). We hypothesized that increased blood pressure in response to dietary Mg2+-depletion leads to increased NLRP3, IL-1ß, and IsoLG production in antigen presenting cells. We found that a Mg2+-depleted diet (0.01% Mg2+ diet) increased blood pressure in mice compared to mice fed a 0.08% Mg2+ diet. Mg2+-depleted mice did not exhibit an increase in total body fluid, as measured by quantitative magnetic resonance. Plasma IL-1ß concentrations were increased (0.13 ± 0.02 pg/mL vs. 0.04 ± 0.02 pg/mL). Using flow cytometry, we observed increased NLRP3 and IL-1ß expression in antigen-presenting cells from spleen, kidney, and aorta. We also observed increased IsoLG production in antigen-presenting cells from these organs. Primary culture of CD11c+ dendritic cells confirmed that low extracellular Mg2+ exerts a direct effect on these cells, stimulating IL-1ß and IL-18 production. The present findings show that NLRP3 inflammasome activation and IsoLG-adduct formation are stimulated when dietary Mg2+ is depleted. Interventions and increased dietary Mg2+ consumption may prove beneficial in decreasing the prevalence of hypertension and cardiovascular disease.
RESUMEN
Toxic levels of trace metals from human activities accumulate in natural environments, yet these metal mixtures are rarely characterized or quantified. Metal mixtures accumulate in historically industrial urban areas and change as economies shift. Previous research has often focused on the sources and fate of a specific element, which limits our understanding of metal contaminant interactions in our environment. Here, we reconstruct the history of metal contamination in a small pond downstream of an interstate highway and downwind of fossil fuel and metallurgical industries that have been active since the middle of the nineteenth century. Metal contamination histories were reconstructed from the sediment record using metal ratio mixing analysis to attribute the relative contributions of contamination sources. Cadmium, copper, and zinc concentrations in sediments accumulated since the construction of major road arteries in the 1930s and 40s are, respectively, 3.9, 2.4, and 6.6 times more concentrated than those during industry-dominated time periods. Shifts in elemental ratios suggest these changes in metal concentrations coincide with increased contributions from road and parking lot traffic, and to a lesser extent, from airborne sources. The metal mixture analysis demonstrates that in near-road environments, contributions from modern surface water pathways can obscure historical atmospheric industrial inputs.
RESUMEN
Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.
Asunto(s)
Calcio , Mucina-1 , Canales Catiónicos TRPV , Animales , Femenino , Humanos , Ratones , Calcio/sangre , Calcio/metabolismo , Calcio/orina , Membrana Celular/metabolismo , Células Cultivadas , Mucina-1/genética , Mucina-1/metabolismo , Canales Catiónicos TRPV/metabolismo , Células Epiteliales/metabolismo , Factores Sexuales , Mutación , Transporte de Proteínas/genéticaRESUMEN
Activation of scramblases is one of the mechanisms that regulates the exposure of phosphatidylserine to the cell surface, a process that plays an important role in tumour immunosuppression. Here we show that chemotherapeutic agents induce overexpression of Xkr8, a scramblase activated during apoptosis, at the transcriptional level in cancer cells, both in vitro and in vivo. Based on this finding, we developed a nanocarrier for co-delivery of Xkr8 short interfering RNA and the FuOXP prodrug to tumours. Intravenous injection of our nanocarrier led to significant inhibition of tumour growth in colon and pancreatic cancer models along with increased antitumour immune response. Targeting Xkr8 in combination with chemotherapy may represent a novel strategy for the treatment of various types of cancers.
Asunto(s)
Nanopartículas , Neoplasias Pancreáticas , Humanos , ARN Interferente Pequeño/uso terapéutico , Apoptosis , Membrana Celular/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Línea Celular Tumoral , Proteínas de la Membrana/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Catchment discretization plays a key role in constructing stormwater models. Traditional methods usually require aerial or topographic data to manually partition the catchment, but this approach is challenging in areas with poor data access. Here, we propose an alternative approach, by drawing Thiessen polygons around sewer nodes to construct a sewershed model. The utility of this approach is evaluated using the EPA's Storm Water Management Model (SWMM) to simulate pipe flow in a sewershed in the City of Pittsburgh. Parameter sensitivities and model uncertainties were explored via Monte Carlo simulations and a simple algorithm applied to calibrate the model. The calibrated model could reliably simulate pipe flow, with a Nash-Sutcliffe efficiency (NSE) of 0.82 when compared to measured flow. The potential influence of sewer data availability on model performance was tested as a function of the number of nodes used to build the model. No statistical differences were observed in model performance when randomly reducing the number of nodes used to build the model (up to 40%). Based on our analyses, the Thiessen polygon approach can be used to construct urban stormwater models and generate good pipe flow simulations even for sewer data limited scenarios.
Asunto(s)
Modelos Teóricos , Lluvia , Movimientos del Agua , Ciudades , AlgoritmosRESUMEN
Medical textiles are subject to particularly harsh disinfection procedures in healthcare settings where exposure risks are high. This work demonstrates a fabric treatment consisting of a reactive silver ink and low surface energy PDMS polymer that provides for superhydrophobicity and antiviral properties against enveloped herpes simplex virus stocks even after extended ultrasonic bleach washing. The antiviral properties of reactive silver ink has not been previously reported or compared with silver nanoparticles. The fabric treatment exhibits high static contact angles and low contact angle hysteresis with water, even after 300 minutes of ultrasonic bleach washing. Similarly, after this bleach washing treatment, the fabric treatment shows reductions of infectious virus quantities by about 2 logs compared to controls for enveloped viruses. The use of silver ink provides for better antiviral efficacy and durability compared to silver nanoparticles due to the use of reactive ionic silver, which demonstrates more conformal coverage of fabric microfibers and better adhesion. This study provides insights for improving the wash durability of antiviral silver fabric treatments and demonstrates a bleach wash durable, repellent antiviral treatment for reusable, functional personal protective equipment applications.
Asunto(s)
Antiinfecciosos , Nanopartículas del Metal , Antivirales , Ácido Hipocloroso , Tinta , Plata/farmacología , Compuestos de Sodio , Textiles , UltrasonidoRESUMEN
Contaminated legacy sediments contribute to modern pollution loadings, particularly trace metals. These contributions are challenging to quantify as metal histories reconstructed from sediment records cannot be easily divided into legacy and concurrent contamination. In particular, the contribution from re-mobilization and delivery of legacy metals stored in catchment soil, colluvial, and fluvial environments are rarely considered or quantified when interpreting sediment records. Here, extended records of metals accumulation for a set of three lakes in Yunnan, China are compared with endmember chemistries using Monte Carlo-Markov Chain mixing models to help identify source contributions to the sediments. This approach allows attribution of metals transported by atmospheric and fluvial mechanisms in a region with a history of mining and metallurgy spanning millennia. These analyses reveal distinct source mixtures and demonstrate the sensitivity of lake records to basin sediment dynamics. In particular, substantial proportions of elevated metal concentrations in these lake systems seem to arise from soil contributions more than from atmospheric deposition of smelting emissions. The largest soil contributions seem to be in Erhai, a lake with erosion prone soils closely "connected" to the lake. Moreover, these invesigations illustrate the potential for mixing approaches to accommodate and clarify uncertainties in metal source and extraction as differences in extraction efficiency can be incorporated into source uncertainty estimates. Ultimately, these approaches emphasize the need to account for fluvial metal transport in interpretation of sediment histories.
Asunto(s)
Metales Pesados , Contaminantes Químicos del Agua , China , Monitoreo del Ambiente , Sedimentos Geológicos , Lagos/análisis , Metales Pesados/análisis , Contaminantes Químicos del Agua/análisisRESUMEN
The molybdopterin enzyme family catalyzes a variety of substrates and plays a critical role in the cycling of carbon, nitrogen, arsenic, and selenium. The dimethyl sulfoxide reductase (DMSOR) subfamily is the most diverse family of molybdopterin enzymes and the members of this family catalyze a myriad of reactions that are important in microbial life processes. Enzymes in the DMSOR family can transform multiple substrates; however, quantitative information about the substrate preference is sparse, and, more importantly, the reasons for the substrate selectivity are not clear. Molybdenum coordination has long been proposed to impact the catalytic activity of the enzyme. Specifically, the molybdenum-coordinating residue may tune substrate preference. As such, molybdopterin enzyme periplasmic nitrate reductase (Nap) is utilized as a vehicle to understand the substrate preference and delineate the kinetic underpinning of the differences imposed by exchanging the molybdenum ligands. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic properties of these variants are discussed and compared with those of the native enzyme, providing quantitative information to understand the function of the molybdenum-coordinating residue.
Asunto(s)
Dimetilsulfóxido/química , Metilaminas/química , Nitrato-Reductasa/química , Nitratos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Campylobacter jejuni/enzimología , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Molibdeno/química , Mutagénesis Sitio-Dirigida , Mutación , Nitrato-Reductasa/genética , Oxidación-Reducción , Periplasma/enzimología , Especificidad por SustratoRESUMEN
One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.
Asunto(s)
Fertilización , Oocitos/metabolismo , Zinc/metabolismo , Ambystoma mexicanum , Animales , Femenino , Hidrozoos , Masculino , Strongylocentrotus purpuratus , Xenopus laevis , Pez CebraRESUMEN
Infiltration-based green infrastructure has become a popular means of reducing stormwater hazards in urban areas. However, the long-term effects of green infrastructure on the geochemistry of roadside environments are poorly defined, particularly given the considerable roadside legacy metal contamination from historic industrial activity and vehicle emissions (e.g., Pb). Most current research on green infrastructure geochemistry is restricted to time periods of less than a year or limited sets of chemical species. This further limits our understanding of systems that evolve over time and are subject to seasonal variability. Between 2016 and 2018, two infiltration trenches in Pittsburgh, PA, were monitored to determine infiltration rates and dissolved nutrient and metal content. The trench water was analyzed to characterize seasonal patterns in both trench function and chemistry. Shifting patterns in infiltration rate and geochemical activity show trends corresponding with seasonal changes. Trench function is dependent on the local water table, with the highest infiltration rates occurring when evapotranspiration is active and groundwater elevation is low. Two seasonal chemical patterns were identified. The first is driven by road salt application in the winter and interaction of the salt pulse increase Pb and Cu concentrations. The second is driven by the formation of summer reducing environments that increase dissolved Fe and Mn. These findings suggest that chemical and hydrological activity in infiltration-based green infrastructure varies seasonally and may remobilize legacy contamination.
RESUMEN
Unconventional natural gas extraction by hydraulic fracturing requires millions of gallons of water and generates flowback water, produced water and recycled fluids of varying chemical composition. Ion chromatography (IC) is a relatively low cost and efficient means to determine the anionic composition, however, the wide range in anionic content of these fluids poses a challenge to analytical methods developed for "natural" waters. We report here that the combination of UV and conductivity detectors increased detection sensitivity (e.g., 10-50 ppb) and expanded the number of anions detectable in a single sample run. Samples from four unconventional shale gas wells, two impoundments, nine conventional oil wells, two freshwater streams and mine drainage samples were analyzed in this study. All produced water samples and impoundment samples had high chloride (17,500-103,000 mg L-1, 93,900 to 134,000 mg L-1, 27,700 and 30,700 mg L-1), bromide (178-996 mg L-1, 183-439 mg L-1, 230 and 260 mg L-1) and conductivity (38,500-160,000 µS/cm3, 95,300 to 183,000 µS/cm3, 61,500 and 103,000 µS/cm3), respectively, relative to mine drainage and freshwater stream samples. Molar ratio analysis using Cl-/Br- to Cl- and SO42-/Cl- to Br- revealed significant differences between the samples, providing a simple means for distinguishing water impacted by different sources of contamination.
Asunto(s)
Aniones/análisis , Industria del Petróleo y Gas/métodos , Sales (Química)/química , Aguas Residuales/química , Contaminantes Químicos del Agua/análisis , Cromatografía , Conductividad Eléctrica , Agua Dulce/química , Yacimiento de Petróleo y GasRESUMEN
Flowback and produced water generated by the hydraulic fracturing of unconventional oil and gas plays contain a suite of cations (e.g., metals) typically in a high salt (e.g., NaCl) matrix. Here, we analyzed the chemical (cation) composition of production fluids associated with natural gas and oil development (e.g., flowback, produced water, impoundment fluids), along with mine drainage, and surface and ground water samples using ICP-OES and ICP-MS. ICP-MS and ICP-OES analytical performance and interference effects were evaluated. Both platforms exhibited excellent analytical spike recoveries, detection limits for blank and spiked solutions, and accuracy for standard certified reference materials. Mass ratio analyses using Ca/Sr, Ca/Mg, Ba/Sr, Mg/Sr, and B and Li, were assessed for their efficacy in differentiation among brines from conventional oil wells, produced water from unconventional oil and gas wells and impoundments, mine drainage treatment pond water, groundwater, and surface water. Examination of Mg/Sr ratios when compared with Li concentrations provide clear separation among the different types of samples, while Ca/Mg versus Ca/Sr correlations were useful for distinguishing between conventional and unconventional oil and gas fluids.
Asunto(s)
Cationes/análisis , Industria del Petróleo y Gas/métodos , Sales (Química)/química , Aguas Residuales/química , Contaminantes Químicos del Agua/análisis , Agua Subterránea/química , Límite de Detección , Metales/análisis , Yacimiento de Petróleo y GasRESUMEN
Identifying the types of contamination and their sources in surface and groundwater is fundamental for effective protection of private and public source waters. Here we employed mass ratio analyses of a variety of anion and cation pairs to characterize flowback, produced water, and mine drainage. These endmembers were used to evaluate the source contributions of natural surface and ground water samples. A total of 1,177 ground water and surface water samples were analyzed including high-quality source waters and waters suspected of being impacted by drilling and mining activity. We found the following chemical ratios resolved different sources of contamination: Mg/Na vs SO4/Cl; SO4/Cl vs Mg/Li; Br/SO4 vs Ba/Cl; and Br vs Mg/Li. While no single parameter or mass ratio pairing by itself was definitive it was possible to converge on a likely source of contamination using multiple lines of analytical evidence. Further, this process clarified sources in impacted samples where one or more parameters commonly considered diagnostic of specific sources (e.g., Br, Ba), were below detection limits (e.g., too dilute) or not tested for. Ultimately, movement of sample values within the mass ratio space allows tracking of changes in water quality and contamination source dynamics as the water chemistry evolves.
Asunto(s)
Aniones/análisis , Cationes/análisis , Monitoreo del Ambiente/métodos , Sales (Química)/química , Contaminantes Químicos del Agua/análisis , Agua Subterránea/química , Gas Natural , Yacimiento de Petróleo y Gas , Industria del Petróleo y Gas , Aguas Residuales/químicaRESUMEN
Fine particulate matter (PM2.5) air pollution varies spatially and temporally in concentration and composition and has been shown to cause or exacerbate adverse effects on human and ecological health. Biomonitoring using airborne tree leaf deposition as a proxy for particulate matter (PM) pollution has been explored using a variety of study designs, tree species, sampling strategies, and analytical methods. In the USA, relatively few have applied these methods using co-located fine particulate measurements for comparison and relying on one tree species with extensive spatial coverage, to capture spatial variation in ambient air pollution across an urban area. Here, we evaluate the utility of this approach, using a spatial saturation design and pairing tree leaf samples with filter-based PM2.5 across Pittsburgh, Pennsylvania, with the goal of distinguishing mobile and stationary sources using PM2.5 composition. Co-located filter and leaf-based measurements revealed some significant associations with traffic and roadway proximity indicators. We compared filter and leaf samples with differing protection from the elements (e.g., meteorology) and PM collection time, which may account for some variance in PM source and/or particle size capture between samples. To our knowledge, this study is among the first to use deciduous tree leaves from a single tree species as biomonitors for urban PM2.5 pollution in the northeastern USA.
Asunto(s)
Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente/métodos , Material Particulado/análisis , Hojas de la Planta/química , Contaminación del Aire/análisis , Humanos , Tamaño de la Partícula , Pennsylvania , ÁrbolesRESUMEN
The large increase in unconventional shale gas extraction has raised concerns about potential water contamination from leaks and spills. Shale gas produced water is challenging to detect in areas impacted by legacy contamination, particularly from conventional sources. Previous studies have proposed combinations of Br, SO4, Ba, Cl, and other more specialized stable isotope systems to delineate shale gas produced water from 1) non-impacted waters and 2) other sources of water contamination. In general, the efforts that rely on relatively simple chemistry do not allow differentiation between conventional and unconventional brine chemistry. We examined variations in Ca/Mg and Ca/Sr ratios that seem to arise from variation in temperature with depth, to differentiate among conventional brines, unconventional brines, and non-impacted waters. This approach was applied to four sedimentary basins in the USGS produced water database: Williston, Michigan, Appalachian, and the Green River basin. In addition, the utility of the system was demonstrated with field samples taken during periods of known unconventional brine releases to surface waters. The Ca/Mg and Ca/Sr ratios allow distinction among these three water types in all basins, suggesting a relatively simple and direct way to evaluate water chemistries in landscapes dominated by unconventional shale gas extraction.
RESUMEN
Mobilization of trace metals from soils to surface waters can impact both human and ecosystem health. This study resamples a water sample archive to explore the spatial pattern of streamwater total concentrations of arsenic, cadmium, copper, lead, and zinc and their associations with biogeochemical controls in northern New England. Road deicing appears to result in elevated trace metal concentrations, as trace metal concentrations are strongly related to sodium concentrations and are most elevated when the sodium: chloride ratio is near 1.0 (~halite). Our results are consistent with previous laboratory and field studies that indicate cation exchange as a metal mobilization mechanism when road salt is applied to soils containing metals. This study also documents associations among sodium, chloride, dissolved organic carbon, iron, and metal concentrations, suggesting cation exchange mechanisms related to road deicing are not the only mechanisms that increase trace metal concentrations in surface waters. In addition to cation exchange, this study considers dissolved organic carbon complexation and oxidation-reduction conditions affecting metal mobility from soils in a salt-rich environment. These observations demonstrate that road deicing has the potential to increase streamwater trace metal concentrations across broad spatial scales and increase risks to human and ecosystem health.
Asunto(s)
Monitoreo del Ambiente , Metales/análisis , Ríos/química , Cloruro de Sodio/farmacología , Oligoelementos/análisis , Transportes , Ecosistema , Humanos , Hielo , Vehículos a Motor , New England , Salinidad , Cloruro de Sodio/análisis , Análisis EspacialRESUMEN
Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 µM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.
Asunto(s)
Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Campylobacter jejuni/enzimología , Clonación Molecular , Nitrato-Reductasa/química , Nitrato-Reductasa/genética , Periplasma/enzimología , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/química , Campylobacter jejuni/genética , Campylobacter jejuni/crecimiento & desarrollo , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Nitrato-Reductasa/metabolismo , Nitratos/química , Nitratos/metabolismo , Periplasma/química , Periplasma/genéticaRESUMEN
Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.