RESUMEN
Seabird guano enters coastal waters providing bioavailable substrates for microbial plankton, but their role in marine ecosystem functioning remains poorly understood. Two concentrations of the water soluble fraction (WSF) of gull guano were added to different natural microbial communities collected in surface waters from the Ría de Vigo (NW Spain) in spring, summer, and winter. Samples were incubated with or without antibiotics (to block bacterial activity) to test whether gull guano stimulated phytoplankton and bacterial growth, caused changes in taxonomic composition, and altered phytoplankton-bacteria interactions. Alteromonadales, Sphingobacteriales, Verrucomicrobia and diatoms were generally stimulated by guano. Chlorophyll a (Chl a) concentration and bacterial abundance significantly increased after additions independently of the initial ambient nutrient concentrations. Our study demonstrates, for the first time, that the addition of guano altered the phytoplankton-bacteria interaction index from neutral (i.e. phytoplankton growth was not affected by bacterial activity) to positive (i.e. phytoplankton growth was stimulated by bacterial activity) in the low-nutrient environment occurring in spring. In contrast, when environmental nutrient concentrations were high, the interaction index changed from positive to neutral after guano additions, suggesting the presence of some secondary metabolite in the guano that is needed for phytoplankton growth, which would otherwise be supplied by bacteria.
Asunto(s)
Ecosistema , Fitoplancton , Animales , Fitoplancton/metabolismo , Clorofila A/metabolismo , Bacterias , AvesRESUMEN
BACKGROUND: Biologics have revolutionized the treatment of many diseases. In this regard, omalizumab (OMA), an anti-IgE monoclonal antibody, is the recommended therapeutic option for patients with chronic spontaneous urticaria (CSU) refractory to second-generation H1-antihistamines. Several studies confirm the efficacy and safety of the drug. However, the literature focusing on the elderly population is scarce, as this age group is often excluded from clinical trials. Therefore, the pharmacological treatment of CSU in elderly patients is a challenge that is increased by their comorbidities and consequent polypharmacy. OBJECTIVES: We describe the real-life safety profile of OMA in elderly patients (≥70 years) with CSU and chronic inducible urticaria (CIndU). We aimed to provide data for daily clinical practice in this vulnerable patient group. METHOD: A retrospective review was performed of the records of patients with CSU/CIndU from May 2003 to December 2019 in the Hospital Universitario La Paz. We describe qualitative and quantitative data according to measures of central tendency. Comparisons between qualitative and quantitative data were performed with the Mann-Whitney U test and the Fisher's test for qualitative variables. A p value <0.05 was considered statistically significant. RESULTS AND CONCLUSIONS: Eighty-nine patients were included, divided into two groups (<70 vs. ≥70 years). The overall rate of adverse events (AEs) was 48%, mainly mild. No association between age and AE was found (p = 0.789). No serious AE such as anaphylaxis was detected. CSU predominated in both groups. CIndU was less prevalent in the elderly (p = 0.017). There was no association between age and the other variables. Although the frequency of neoplasms was slightly higher in the elderly with OMA, we found no difference compared to the incidence of neoplasms in the general population. Therefore, our data suggest that OMA may be a safe treatment in elderly people with CSU/CIndU for prolonged periods of treatment, although further studies with larger samples are needed to corroborate our observations.
Asunto(s)
Antialérgicos , Urticaria Crónica , Neoplasias , Urticaria , Humanos , Anciano , Omalizumab/uso terapéutico , Antialérgicos/efectos adversos , Urticaria/tratamiento farmacológico , Urticaria/epidemiología , Enfermedad Crónica , Urticaria Crónica/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Urticaria Crónica Inducible , Neoplasias/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Biofouling is the major factor that limits long-term monitoring studies with automated optical instruments. Protection of the sensing areas, surfaces, and structural housing of the sensors must be considered to deliver reliable data without the need for cleaning or maintenance. In this work, we present the design and field validation of different techniques for biofouling protection based on different housing materials, biocides, and transparent coatings. Six optical turbidity probes were built using polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), PLA with copper filament, ABS coated with PDMS, ABS coated with epoxy and ABS assembled with a system for in situ chlorine production. The probes were deployed in the sea for 48 days and their anti-biofouling efficiency was evaluated using the results of the field experiment, visual inspections, and calibration signal loss after the tests. The PLA and ABS were used as samplers without fouling protection. The probe with chlorine production outperformed the other techniques, providing reliable data during the in situ experiment. The copper probe had lower performance but still retarded the biological growth. The techniques based on transparent coatings, epoxy, and PDMS did not prevent biofilm formation and suffered mostly from micro-biofouling.
Asunto(s)
Incrustaciones Biológicas , Desinfectantes , Biopelículas , Cloro , Cobre/química , Incrustaciones Biológicas/prevención & control , ClorurosRESUMEN
OBJECTIVE: To assess the safety of biological therapy for severe T2 asthma (omalizumab, mepolizumab, benralizumab and reslizumab) under real-life conditions in elderly patients older than 70 years. METHODS: Retrospective data collection including clinical characteristics, comorbidities, treatment, disease control and adverse events (AE) of all patients with severe asthma on biological therapy older than 70 years seen in the Severe Asthma Unit of our hospital. RESULTS: Of 147 patients with severe asthma being treated with biologics, 21 patients older than 70 years were included. The median age of these patients was 76.3 years (range 71-86) and the majority were women (n = 18, 85.7%). There were 9 patients (42.9%) who experienced an AE related to biological treatment. Four (44.4%) were in treatment with omalizumab, two (22.2%) with mepolizumab, two patients (22.2%) with reslizumab and one (11.1%) with benralizumab. The median FEV1 (%) was 66%. These patients had a considerably higher body mass index (BMI). No significant differences were found for any other variable. Most of the AE reported were considered mild with the exception of one case of systemic AE (anaphylaxis) associated with omalizumab. CONCLUSION: This study indicates that the prescription of biological therapy in elderly patients with severe asthma seems to be safe. More evidence is needed in this particular population.
Asunto(s)
Antiasmáticos , Asma , Productos Biológicos , Anciano , Anciano de 80 o más Años , Antiasmáticos/efectos adversos , Asma/terapia , Productos Biológicos/efectos adversos , Terapia Biológica , Femenino , Humanos , Masculino , Omalizumab/efectos adversos , Estudios RetrospectivosRESUMEN
Nitrous oxide (N2O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N2O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N2O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N2O and CO2 in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N2O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N2O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N2O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO2 emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO2 fixation and release.
Asunto(s)
Chlamydomonas reinhardtii , Gases de Efecto Invernadero , Microalgas , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Microalgas/metabolismo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Óxido Nitroso/metabolismoRESUMEN
Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na+/K+ is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na+ transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species.
Asunto(s)
Proteínas de Transporte de Catión , Solanum lycopersicum , Solanum , Proteínas de Transporte de Catión/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Fitomejoramiento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Potasio/metabolismo , Salinidad , Sodio/metabolismo , Solanum/genéticaRESUMEN
Cobalamin (B12) is an essential cofactor that is exclusively synthesized by some prokaryotes while many prokaryotes and eukaryotes require an external supply of B12. The spatial and temporal availability of B12 is poorly understood in marine ecosystems. Field measurements of B12 along with a large set of ancillary biotic and abiotic factors were obtained during three oceanographic cruises in the NW Iberian Peninsula, covering different spatial and temporal scales. B12 concentrations were remarkably low (<1.5 pM) in all samples, being significantly higher at the subsurface Eastern North Atlantic Central Water than at shallower depths, suggesting that B12 supply in this water mass is greater than demand. Multiple regression models excluded B12 concentration as predictive variable for phytoplankton biomass or production, regardless of the presence of B12-requiring algae. Prokaryote production was the best predictor for primary production, and eukaryote community composition was better correlated with prokaryote community composition than with nutritional resources, suggesting that biotic interactions play a significant role in regulating microbial communities. Interestingly, co-occurrence network analyses based on 16S and 18S rRNA sequences allowed the identification of significant associations between potential B12 producers and consumers (e.g. Thaumarchaeota and Dynophyceae, or Amylibacter and Ostreococcus respectively), which can now be investigated using model systems in the laboratory.
Asunto(s)
Microbiota , Plancton , Océano Atlántico , Plancton/genética , Agua de Mar , Vitamina B 12RESUMEN
B vitamins are essential cofactors for practically all living organisms on Earth and are produced by a selection of microorganisms. An imbalance between high demand and limited production, in concert with abiotic processes, may explain the low availability of these vitamins in marine systems. Natural microbial communities from surface shelf water in the productive area off northwestern Spain were enclosed in mesocosms in winter, spring, and summer 2016. In order to explore the impact of B-vitamin availability on microbial community composition (16S and 18S rRNA gene sequence analysis) and bacterial function (metatranscriptomics analysis) in different seasons, enrichment experiments were conducted with seawater from the mesocosms. Our findings revealed that significant increases in phytoplankton or prokaryote biomass associated with vitamin B12 and/or B1 amendments were not accompanied by significant changes in community composition, suggesting that most of the microbial taxa benefited from the external B-vitamin supply. Metatranscriptome analysis suggested that many bacteria were potential consumers of vitamins B12 and B1, although the relative abundance of reads related to synthesis was ca. 3.6-fold higher than that related to uptake. Alteromonadales and Oceanospirillales accounted for important portions of vitamin B1 and B12 synthesis gene transcription, despite accounting for only minor portions of the bacterial community. Flavobacteriales appeared to be involved mostly in vitamin B12 and B1 uptake, and Pelagibacterales expressed genes involved in vitamin B1 uptake. Interestingly, the relative expression of vitamin B12 and B1 synthesis genes among bacteria strongly increased upon inorganic nutrient amendment. Collectively, these findings suggest that upwelling events intermittently occurring during spring and summer in productive ecosystems may ensure an adequate production of these cofactors to sustain high levels of phytoplankton growth and biomass. IMPORTANCE B vitamins are essential growth factors for practically all living organisms on Earth that are produced by a selection of microorganisms. An imbalance between high demand and limited production may explain the low concentration of these compounds in marine systems. In order to explore the impact of B-vitamin availability on bacteria and algae in the coastal waters off northwestern Spain, six experiments were conducted with natural surface water enclosed in winter, spring, and summer. Our findings revealed that increases in phytoplankton or bacterial growth associated with B12 and/or B1 amendments were not accompanied by significant changes in community composition, suggesting that most microorganisms benefited from the B-vitamin supply. Our analyses confirmed the role of many bacteria as consumers of vitamins B12 and B1, although the relative abundance of genes related to synthesis was ca. 3.6-fold higher than that related to uptake. Interestingly, prokaryote expression of B12 and B1 synthesis genes strongly increased when inorganic nutrients were added. Collectively, these findings suggest that upwelling of cold and nutrient-rich waters occurring during spring and summer in this coastal area may ensure an adequate production of B vitamins to sustain high levels of algae growth and biomass.
Asunto(s)
Microbiota , Agua de Mar/microbiología , Tiamina , Vitamina B 12 , Complejo Vitamínico B , Océano Atlántico , Plancton , España , TranscriptomaRESUMEN
Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO43- ; 10 mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO43- and stored polyP within minutes of adding PO43- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO43- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO43- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO43- assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO43- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO43- to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO43- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.
Asunto(s)
Chlamydomonas reinhardtii , Transporte Biológico , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chaperonas Moleculares/metabolismo , Fósforo , PolifosfatosRESUMEN
The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by three proteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonas reinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonas reinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Sistema de Señalización de MAP Quinasas , Nitrógeno/metabolismo , Proteínas Algáceas/genética , Compuestos de Amonio/metabolismo , Chlamydomonas reinhardtii/genética , Regulación de la Expresión Génica de las Plantas , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/genéticaRESUMEN
Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.
Asunto(s)
Microalgas/genética , Plásmidos/genética , Transgenes/genética , Antibacterianos/farmacología , Marcadores Genéticos/genética , Kanamicina Quinasa/genética , Paromomicina/farmacología , Regiones Promotoras Genéticas/genética , Streptomyces/efectos de los fármacos , Streptomyces/genética , Transformación Genética/genéticaRESUMEN
Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Óxido Nitroso/metabolismo , Chlamydomonas reinhardtii/genética , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
In this paper, we have studied the chiroptical properties of a family of o-oligo(phenyleneethynylene) (o-OPE) derivatives with different steric hindrance. Experimental results show high dissymmetry factors (gabs and glum up to 1.1 × 10-2 ) and very similar electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) for all the derivatives that make this basic o-OPE scaffold a robust pure organic emitter. Vibrational circular dichroism spectra are used to characterize conformational properties in solution. Density functional theory and time-dependent density functional theory calculations support experimental results also proving that ECD and CPL are almost exclusively linked to helical moiety and not to size or conformation of substituents. As chiroptical properties of these emitters are independent of substituents, this OPE scaffold can be used as basic skeleton for the design of sensing probes with high CPL efficiencies.
RESUMEN
All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue.
Asunto(s)
Coenzimas/metabolismo , Enzimas/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Animales , Miosinas Cardíacas/metabolismo , Coenzimas/biosíntesis , Enzimas/química , Enzimas/genética , Células Eucariotas/metabolismo , Mamíferos , Redes y Vías Metabólicas , Metaloproteínas/biosíntesis , Molibdeno/metabolismo , Cofactores de Molibdeno , Cadenas Ligeras de Miosina/metabolismo , Nitrato-Reductasa/metabolismo , Nitritos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
The green alga Chlamydomonas is a valuable model system capable of assimilating different forms of nitrogen (N). Nitrate (NO3-) has a relevant role in plant-like organisms, first as a nitrogen source for growth and second as a signalling molecule. Several modules are necessary for Chlamydomonas to handle nitrate, including transporters, nitrate reductase (NR), nitrite reductase (NiR), GS/GOGAT enzymes for ammonium assimilation, and regulatory protein(s). Transporters provide a first step for influx/efflux, homeostasis, and sensing of nitrate; and NIT2 is the key transcription factor (RWP-RK) for mediating the nitrate-dependent activation of a number of genes. Here, we review how NR participates in the cycle NO3- âNO2- âNO âNO3-. NR uses the partner protein amidoxime-reducing component/nitric oxide-forming nitrite reductase (ARC/NOFNiR) for the conversion of nitrite (NO2-) into nitric oxide (NO). It also uses the truncated haemoglobin THB1 in the conversion of nitric oxide to nitrate. Nitric oxide is a negative signal for nitrate assimilation; it inhibits the activity and expression of high-affinity nitrate/nitrite transporters and NR. During this cycle, the positive signal of nitrate is transformed into the negative signal of nitric oxide, which can then be converted back into nitrate. Thus, NR is back in the spotlight as a strategic regulator of the nitric oxide cycle and the nitrate assimilation pathway.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas/metabolismo , Nitrato-Reductasa/metabolismo , Ciclo del Nitrógeno , Óxido Nítrico/metabolismo , Nitritos/metabolismoRESUMEN
Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod's surface. The model sensors were further characterized by measuring their dose-response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response.
RESUMEN
The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of several N-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5 (crCytb5-1) and a cytochrome b5 reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction with its partners and the function of important conserved amino acids. Interactions among crARC, crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein aggregation or oligomerization.
Asunto(s)
Coenzimas/metabolismo , Citocromos b5/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/metabolismo , Coenzimas/química , Coenzimas/genética , Citocromos b5/química , Citocromos b5/genética , Metaloproteínas/química , Metaloproteínas/genética , Cofactores de Molibdeno , Unión Proteica , Multimerización de Proteína , Pteridinas/químicaRESUMEN
Hemoglobins are ubiquitous proteins that sense, store and transport oxygen, but the physiological processes in which they are implicated is currently expanding. Recent examples of previously unknown hemoglobin functions, which include scavenging of the signaling molecule nitric oxide (NO), illustrate how the implication of hemoglobins in different cell signaling processes is only starting to be unraveled. The extent and diversity of the hemoglobin protein family suggest that hemoglobins have diverged and have potentially evolved specialized functions in certain organisms. A unique model organism to study this functional diversity at the cellular level is the green alga Chlamydomonas reinhardtii because, among other reasons, it contains an unusually high number of a particular type of hemoglobins known as truncated hemoglobins (THB1-THB12). Here, we reveal a cell signaling function for a truncated hemoglobin of Chlamydomonas that affects the nitrogen assimilation pathway by simultaneously modulating NO levels and nitrate reductase (NR) activity. First, we found that THB1 and THB2 expression is modulated by the nitrogen source and depends on NIT2, a transcription factor required for nitrate assimilation genes expression. Furthermore, THB1 is highly expressed in the presence of NO and is able to convert NO into nitrate in vitro. Finally, THB1 is maintained on its active and reduced form by NR, and in vivo lower expression of THB1 results in increased NR activity. Thus, THB1 plays a dual role in NO detoxification and in the modulation of NR activity. This mechanism can partly explain how NO inhibits NR post-translationally.
Asunto(s)
Proteínas Algáceas/fisiología , Chlamydomonas reinhardtii/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Nitrato-Reductasa/metabolismo , Óxido Nítrico/metabolismo , Hemoglobinas Truncadas/fisiología , Proteínas Algáceas/química , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Comunicación Celular , Chlamydomonas reinhardtii/genética , Regulación de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMEN
Read the highlighted article 'Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?' on page 823.