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Three kinds of coronaviruses are highly pathogenic to humans, and two of them mainly infect humans through Angiotensin-converting enzyme 2 ï¼ACE2ï¼receptors. Therefore, specifically blocking ACE2 binding at the interface with the receptor-binding domain is promising to achieve both preventive and therapeutic effects of coronaviruses. Alternatively, drug-targeted delivery based on ACE2 receptors can further improve the efficacy and safety of inhalation drugs. Here, these two approaches are innovatively combined by designing a nanoemulsion (NE) drug delivery system (termed NE-AYQ) for inhalation that targets binding to ACE2 receptors. This inhalation-delivered remdesivir nanoemulsion (termed RDSV-NE-AYQ) effectively inhibits the infection of target cells by both wild-type and mutant viruses. The RDSV-NE-AYQ strongly inhibits Severe acute respiratory syndrome coronavirus 2 at two dimensions: they not only block the binding of the virus to host cells at the cell surface but also restrict virus replication intracellularly. Furthermore, in the mouse model of acute lung injury, the inhaled drug delivery system loaded with anti-inflammatory drugs (TPCA-1-NE-AYQ) can significantly alleviate the lung tissue injury of mice. This smart combination provides a new choice for dealing with possible emergencies in the future and for the rapid development of inhaled drugs for the treatment of respiratory diseases.
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COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratones , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Peptidil-Dipeptidasa A/farmacología , Replicación ViralRESUMEN
Brassinosteroids (BRs) play critical roles in plant growth and development and regulate many important agronomic traits. However, the functions of BRs in strawberry are unclear. This study identified two mutants, named P6 and R87, in woodland strawberry (Fragaria vesca) from EMS mutagenesis populations that exhibit narrow leaves, petals and sepals. Mapping by sequencing and genetic studies revealed that the F. vesca CYP734A129, encoding a putative BR catabolic enzyme, is the causative gene for both P6 and R87. Overexpression of CYP734A129 in both F. vesca and Arabidopsis causes a severe dwarf phenotype, and the BRI1-EMS-SUPPRESSOR 1 (BES1) protein is less abundant in the CYP734A129-overexpressing Arabidopsis seedlings. This suggests that CYP734A129 is functionally conserved with CYP734A1, as a BR-inactivating enzyme. Transcriptome analysis of young leaves revealed that four BR biosynthetic genes were significantly downregulated in P6 (cyp734a129), and photosynthesis-related genes were highly enriched among the up-regulated genes in P6 compared to the wild type. This further supports that CYP734A129 inactivates BRs in F. vesca. Furthermore, we showed that mutations in CYP734A129 do not affect fruit shape and color during ripening in strawberry. Overall, our results suggest that F. vesca CYP734A129 is a BR catabolic enzyme, and provide insights into the roles of CYP734A129 in strawberry.
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Arabidopsis , Fragaria , Arabidopsis/genética , Brasinoesteroides/metabolismo , Hojas de la Planta/metabolismo , Desarrollo de la Planta , Regulación de la Expresión Génica de las PlantasRESUMEN
Rationale: Myocardial injury triggers intense oxidative stress, inflammatory response, and cytokine release, which are essential for myocardial repair and remodeling. Excess reactive oxygen species (ROS) scavenging and inflammation elimination have long been considered to reverse myocardial injuries. However, the efficacy of traditional treatments (antioxidant, anti-inflammatory drugs and natural enzymes) is still poor due to their intrinsic defects such as unfavorable pharmacokinetics and bioavailability, low biological stability, and potential side effects. Nanozyme represents a candidate to effectively modulate redox homeostasis for the treatment of ROS related inflammation diseases. Methods: We develop an integrated bimetallic nanozyme derived from metal-organic framework (MOF) to eliminate ROS and alleviate inflammation. The bimetallic nanozyme (Cu-TCPP-Mn) is synthesized by embedding manganese and copper into the porphyrin followed by sonication, which could mimic the cascade activities of superoxide dismutase (SOD) and catalase (CAT) to transform oxygen radicals to hydrogen peroxide, followed by the catalysis of hydrogen peroxide into oxygen and water. Enzyme kinetic analysis and oxygen-production velocities analysis were performed to evaluate the enzymatic activities of Cu-TCPP-Mn. We also established myocardial infarction (MI) and myocardial ischemia-reperfusion (I/R) injury animal models to verify the ROS scavenging and anti-inflammation effect of Cu-TCPP-Mn. Results: As demonstrated by kinetic analysis and oxygen-production velocities analysis, Cu-TCPP-Mn nanozyme possesses good performance in both SOD- and CAT-like activities to achieve synergistic ROS scavenging effect and provide protection for myocardial injury. In both MI and I/R injury animal models, this bimetallic nanozyme represents a promising and reliable technology to protect the heart tissue from oxidative stress and inflammation-induced injury, and enables the myocardial function to recover from otherwise severe damage. Conclusions: This research provides a facile and applicable method to develop a bimetallic MOF nanozyme, which represents a promising alternative to the treatment of myocardial injuries.
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Estructuras Metalorgánicas , Daño por Reperfusión Miocárdica , Animales , Especies Reactivas de Oxígeno , Peróxido de Hidrógeno , Cinética , Superóxido Dismutasa/metabolismo , Oxígeno , CatálisisRESUMEN
The plant-specific transcription factor LEAFY (LFY), generally maintained as a single-copy gene in most angiosperm species, plays critical roles in flower development. The woodland strawberry (Fragaria vesca) possesses four LFY homologs in the genome; however, their respective functions and evolution remain unknown. Here, we identified and validated that mutations in one of the four LFY homologs, FveLFYa, cause homeotic conversion of floral organs and reiterative outgrowth of ectopic flowers. In contrast to FveLFYa, FveLFYb/c/d appear dispensable under normal growth conditions, as fvelfyc mutants are indistinguishable from wild type and FveLFYb and FveLFYd are barely expressed. Transgenic analysis and yeast one-hybrid assay showed that FveLFYa and FveLFYb, but not FveLFYc and FveLFYd, are functionally conserved with AtLFY in Arabidopsis (Arabidopsis thaliana). Unexpectedly, LFY-binding site prediction and yeast one-hybrid assay revealed that the transcriptional links between LFY and the APETALA1 (AP1) promoter/the large AGAMOUS (AG) intron are missing in F. vesca, which is due to the loss of LFY-binding sites. The data indicate that mutations in cis-regulatory elements could contribute to LFY evolution. Moreover, we showed that FveLFYa is involved in leaf development, as approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Phylogenetic analysis indicated that LFY homologs in Fragaria species may arise from recent duplication events in their common ancestor and are undergoing convergent gene loss. Together, these results provide insight into the role of LFY in flower and leaf development in strawberry and have important implications for the evolution of LFY.
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Proteínas de Arabidopsis , Arabidopsis , Fragaria , Fragaria/genética , Fragaria/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Arabidopsis/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Flores , Regulación de la Expresión Génica de las PlantasRESUMEN
Rationale: Conventional 18F-labeling methods that demand substrate pre-modification or lengthy radiosynthesis procedures have impeded the visualization and translation of numerous biomolecules, as biomarkers or ligands, using modern positron emission tomography techniques in vivo. Moreover, 18F-labeled biomolecules in high molar activity (Am) that are indispensable for sensitive imaging could be only achieved under strict labeling conditions. Methods: Herein, 18F-labeled fluorothiophosphate (FTP) synthons in high Am have been generated rapidly in situ in reaction solutions with < 5% water via nucleophilic substitution by wet [18F]F-, which required minimal processing from cyclotron target water. Results: Various 18F-labeled FTP synthons have been prepared in 30 sec at room temperature with high radiochemical yields > 75% (isolated, non-decay-corrected). FTP synthons with unsaturated hydrocarbon or activated ester group can conjugate with typical small molecules, peptides, proteins, and metallic nanoparticles. 337-517 GBq µmol-1 Am has been achieved for 18F-labeled c(RGDyK) peptide using an automatic module with 37-74 GBq initial activity. Conclusion: The combination of high 18F-fluorination efficiency of FTP synthons and following mild conjugation condition provides a universal simplified one-pot 18F-labeling method for broad unmodified biomolecular substrates.
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Radioisótopos de Flúor , Tomografía Computarizada por Rayos X , Radioisótopos de Flúor/química , Tomografía de Emisión de Positrones/métodos , Proteínas , Péptidos/química , Radiofármacos , AguaRESUMEN
The strategy of combining a vaccine with immune checkpoint inhibitors has been widely investigated in cancer management, but the complete response rate for this strategy is still unresolved. We describe a genetically engineered cell membrane nanovesicle that integrates antigen self-presentation and immunosuppression reversal (ASPIRE) for cancer immunotherapy. The ASPIRE nanovaccine is derived from recombinant adenovirus-infected dendritic cells in which specific peptide-major histocompatibility complex class I (pMHC-I), anti-PD1 antibody and B7 co-stimulatory molecules are simultaneously anchored by a programmed process. ASPIRE can markedly improve antigen delivery to lymphoid organs and generate broad-spectrum T-cell responses that eliminate established tumours. This work presents a powerful vaccine formula that can directly activate both native T cells and exhausted T cells, and suggests a general strategy for personalized cancer immunotherapy.
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Vacunas contra el Cáncer , Neoplasias , Presentación de Antígeno , Humanos , Terapia de Inmunosupresión , Inmunoterapia , Neoplasias/terapiaRESUMEN
Photoacoustic imaging (PAI) in the second near-infrared window (NIR-II) is conducive to deep-tissue imaging due to small scattering coefficients, but often requires exogenous imaging agents. At present, nanoparticle-based NIR-II imaging agents are mainly used in non-clinical studies, some basic components of which are resistant to metabolism inâ vivo. The aim of this study was to examine the â¼600â Da croconaine absorber A1094, absorbing lights around 1094â nm, as a rare, small-molecule NIR-II imaging agent inâ vivo. The clinical translational potential of A1094 injection were systematically revealed, including sufficient solubility and freeness in blood, good anti-interference ability, and favourable pH/oxidation-reduction/metabolic stabilities. After intravenous administration of A1094 injection, PAI of murine ears exhibited comparable capillaries visibility to that of PAI with popular Au nanorods. The contrasts achieved with A1094 and Au nanorods were 1.78 and 1.29â times higher than before administration, in the healthy group, and 3.25 and 1.58â times higher in the inflammation group. Notably, A1094 demonstrated a desired faster liver clearance than Au nanorods. The PAI signal of A1094 was cleared by 74.2 % after 3â h, whereas Au nanorods were only cleared by 43.1 %. The main metabolic mechanisms of A1094 were identified as N-methylation and lipid hydrolysis by murine liver microsomes inâ vitro. Therefore, A1094 may have promising clinical potential as a stable and fast-clearing NIR-II imaging agent.
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Materiales Biocompatibles/química , Hígado/diagnóstico por imagen , Técnicas Fotoacústicas , Bibliotecas de Moléculas Pequeñas/química , Animales , Materiales Biocompatibles/metabolismo , Línea Celular , Oro/química , Rayos Infrarrojos , Nanopartículas del Metal/química , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/metabolismo , Espectroscopía Infrarroja CortaRESUMEN
18F-Labelled pyrrolopyrimidines were synthesized and evaluated as positron emission tomography (PET) probes to determine leucine-rich repeat kinase 2 (LRRK2) expression in the brain. With pyrrolopyrimidine derivative PF-06447475 as the lead compound, two in vivo-stable 18F-labelled pyrrolopyrimidines ([18F]1 and [18F]2) were synthesized automatically at radiochemical yields 8-10% (non-decay-corrected) with molar activities of 0.95 and 0.5 GBq/µmol, respectively. The measured Kd of 6.90 nM for 1 and 14.27 nM for 2 demonstrated high affinities for LRRK2. The LRRK2 G2019S mice had higher uptakes (P < 0.01) of [18F]1 in the olfactory bulb, striatum, and hippocampus than WT mice during microPET/CT imaging, consistent with immunohistology results of LRRK2 distribution. [11C]CFT microPET/CT imaging demonstrated a lower expression of dopamine transporter in LRRK2 G2019S mice. Parkinson's disease-like deficits in dopamine transporter synthesis and cognitive declines were noticed along with LRRK2 expression increase in the olfactory bulb, striatum, and hippocampus. Therefore, 18F-labelled pyrrolopyrimidines can reflect real-time LRRK2 expression changes implicated in Parkinson's disease, which paves the way for LRRK2-related neurodegenerative precise therapy.
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Encéfalo/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Sondas Moleculares/química , Enfermedad de Parkinson/metabolismo , Pirimidinas/química , Pirroles/química , Animales , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Radioisótopos de Flúor , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Sondas Moleculares/síntesis química , Estructura Molecular , Enfermedad de Parkinson/patología , Tomografía de Emisión de Positrones , Pirimidinas/síntesis química , Pirroles/síntesis química , Relación Estructura-ActividadRESUMEN
Corneal neovascularization (CNV) is one of the main factors that induce blindness worldwide. However, current medical treatments cannot achieve non-invasive and safe inhibition of CNV. A noninvasive photoacoustic imaging (PAI)-guided method is purposed for the regression of CNV. PAI can monitor the oxygen saturation of cornea blood vessels through the endogenous contrast of hemoglobin and trace administrated drugs by themselves as exogenous contrast agents. An indocyanine green (ICG)-based nanocomposite (R-s-ICG) is prepared for CNV treatment via eye drops and subconjunctival injections. It is demonstrated that R-s-ICG can enrich corneal tissues and pathological blood vessels rapidly with minor residua in normal eyeball tissues. Anti-CNV treatment-driven changes in the blood vessels are assessed by real-time multimodal PAI in vivo, and then a safe laser irradiation strategy through the canthus is developed for phototherapy and gene therapy synergistic treatment. The treatment leads to the efficient inhibition of CNV with faint damages to normal tissues.
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Although emerging evidence suggests that the pathogenesis of Parkinson's disease (PD) is closely related to the aggregation of alpha-synuclein (α-syn) in the midbrain, the clearance of α-syn remains an unmet clinical need. Here, we develop a simple and efficient strategy for fabricating the α-syn nanoscavenger for PD via a reprecipitation self-assembly procedure. The curcumin analogue-based nanoscavenger (NanoCA) is engineered to be capable of a controlled-release property to stimulate nuclear translocation of the major autophagy regulator, transcription factor EB (TFEB), triggering both autophagy and calcium-dependent exosome secretion for the clearance of α-syn. Pretreatment of NanoCA protects cell lines and primary neurons from MPP+-induced neurotoxicity. More importantly, a rapid arousal intranasal delivery system (RA-IDDS) was designed and applied for the brain-targeted delivery of NanoCA, which affords robust neuroprotection against behavioral deficits and promotes clearance of monomer, oligomer, and aggregates of α-syn in the midbrain of an MPTP mouse model of PD. Our findings provide a clinically translatable therapeutic strategy aimed at neuroprotection and disease modification in PD.
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Curcumina/uso terapéutico , Nanoestructuras/química , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Curcumina/síntesis química , Curcumina/química , Liberación de Fármacos , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Células PC12 , Enfermedad de Parkinson/patología , Tamaño de la Partícula , Agregado de Proteínas/efectos de los fármacos , Ratas , Propiedades de Superficie , alfa-Sinucleína/metabolismoRESUMEN
Perihilar cholangiocarcinoma (PHCC) presents a formidable challenge due to its occult anatomic location, aggressive growth, insensitivity to conventional chemotherapy, and poor prognosis. Herein, we engineered a human epidermal growth factor receptor 2 (HER2) affibody to the surface of cell membrane nanovesicles (A-NVs) in a ligand-oriented manner and loaded them with indocyanine green (ICG) as precision theranostics for PHCC treatment. The A-NVs@ICG were prepared and exhibited satisfactory targeting effects in HER2-overexpressing PHCC cells. In vivo fluorescence and photoacoustic imaging demonstrated that A-NVs@ICG promoted the accumulation of ICG in PHCC tissue, leading to enhanced tumor regression and improved anti-cancer effects when combined with photoirradiation. Therefore, bio-engineered A-NVs@ICG represent a promising nanotheranostic agent for PHCC with potential for clinical translation.
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Neoplasias de los Conductos Biliares/terapia , Hipertermia Inducida/métodos , Verde de Indocianina/química , Tumor de Klatskin/terapia , Receptor ErbB-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Neoplasias de los Conductos Biliares/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Tumor de Klatskin/metabolismo , Ratones , Nanopartículas , Trasplante de Neoplasias , Técnicas Fotoacústicas , Medicina de Precisión , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Nanomedicina Teranóstica , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.
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Polinucleotido Adenililtransferasa/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metilación de ADN/genética , Metilación de ADN/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Polen/metabolismo , Polinucleotido Adenililtransferasa/genética , ARN de Planta/genética , ARN de Planta/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
As a carbon free fuel and a hydrogen-energy carrier, ammonia is expected to be one of the promising energy carriers and to be widely used in industries. In this study, the parameters of ammonia/oxygen flame, such as laminar flame speed, the Markstein length, laminar flame thickness, and critical radius of flame instability onset have been investigated experimentally and numerically. A spherical ammonia /oxygen flame propagated in a constant volume chamber and a high speed digital Schilieren photograph system were used in the study. The influences of initial pressure and equivalence ratio on flame propagation have been investigated. It was found that the maximum laminar flame speed in ammonia/oxygen mixture is 1.09 m/s. The flame thickness decreases with the initial pressure increase. Generally, Markstein length increases with the increasing of equivalence ratio while it decreases with the increasing of initial pressure. The minimum critical radius in ammonia/oxygen is 1.8 cm. And the critical radius decreases with the increase of initial pressure. The mechanisms of flame instability in ammonia/oxygen mixture were analyzed, and the stabilize effect of flame stretch in ammonia/oxygen have been observed.
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We report the rational design of coordination-driven self-assembly metal-organic nanostructures for multifunctional nanotheranostics. Zinc(II) coordination-based nano-formulations capable of loading indocyanine green (ICG) and therapeutic genes were prepared to achieve a fluorescence/photoacoustic imaging-guided combination photo/gene therapy strategy. We showed the enhanced theranostic capability of zinc(II)-dipicolylamine-assisted assembly of ICG, as well as simultaneous targeted gene delivery in an experimental mouse model of cancer. Such a co-assembly strategy provides a facile way to achieve combined therapeutic functions for personalized nanomedicine.
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Terapia Genética/métodos , Nanomedicina/métodos , Fototerapia/métodos , Zinc/química , HumanosRESUMEN
As one of the most widely studied epigenetic modifications, DNA methylation has an important influence on human traits and cancers. Dynamic variations in DNA methylation have been reported in malignant neoplasm and aging; however, the mechanisms remain poorly understood. By constructing an age-associated and cancer-related weighted network (ACWN) based on the correlation of the methylation level and the protein-protein interaction, we found that DNA methylation changes associated with age were closely related to the occurrence of cancer. Additional analysis of 102 module genes mined from the ACWN revealed discrimination based on two main patterns. One pattern involved methylation levels that increased with aging and were higher in cancer patients compared with normal controls (HH pattern). The other pattern involved methylation levels that decreased with aging and were lower in cancer compared with normal (LL pattern). Upon incorporation with gene expression levels, 25 genes were filtered based on negative regulation by DNA methylation. These genes were regarded as potential cancer risk markers that were influenced by age in the process of carcinogenesis. Our results will facilitate further studies regarding the impact of the epigenetic effects of aging on diseases and will aid in the development of tailored cancer preventive strategies.
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Envejecimiento/patología , Biomarcadores de Tumor/análisis , Metilación de ADN , ADN/química , Epigénesis Genética , Neoplasias/diagnóstico , Neoplasias/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.
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Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Chlorophyta/metabolismo , Lípidos/biosíntesis , Nitrógeno/metabolismo , Complejos de ATP Sintetasa/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Catalasa/genética , Catalasa/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chlorella/clasificación , Chlorella/genética , Chlorella/metabolismo , Chlorophyta/genética , Transporte de Electrón/genética , Transporte de Electrón/fisiología , Immunoblotting , Microscopía Confocal , Mutación , Fotosíntesis/genética , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema I/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tilacoides/enzimología , Tilacoides/metabolismoRESUMEN
Changes in the cytosolic Ca(2+) levels and the role of Ca(2+) signal transduction in neutral lipid synthesis in Chlorella sp. C2 under nitrogen starvation conditions were investigated. The results detected by using the scanning ion-selective electrode technique demonstrate that nitrogen starvation induced significant Ca(2+) influx across the plasma membrane into cells. Ca(2+) fluorescence imaging and flow cytometry were used to estimate the effect of this Ca(2+) influx on the generation of the Ca(2+) signal, and the results showed that the cytosolic Ca(2+) concentration increased transiently and then remained at a stable, high level when the cells were exposed to nitrogen starvation. However, the increase could be inhibited by pre-treatment with the Ca(2+) channel blockers ruthenium red, verapamil and GdCl3, indicating that both the influx of Ca(2+) from the extracellular space via Ca(2+) channels that are localized in the plasma membrane and the release of Ca(2+) from intracellular calcium storage via the internal calcium store were required for the generation and transduction of the Ca(2+) signal. During nitrogen starvation, neutral lipid synthesis in Chlorella sp. C2 in response to stress conditions was also inhibited to differing degrees by pre-treatment with the three Ca(2+) channel blockers, demonstrating the regulation of Ca(2+) via these Ca(2+) channels in neutral lipid synthesis. The results suggested that by transduction of extracellular stress signals into the cell and the regulation of the Ca(2+) signal in neutral lipid synthesis, Ca(2+) signal transduction played important roles in the response mechanism of Chlorella sp. C2 to nitrogen starvation.
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Calcio/metabolismo , Chlorophyta/metabolismo , Aceites de Plantas/metabolismo , Chlorophyta/efectos de los fármacos , Chlorophyta/genética , Gadolinio/farmacología , Nitrógeno/deficiencia , Rojo de Rutenio/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Verapamilo/farmacologíaRESUMEN
Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H2O2 treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.