RESUMEN
The insect cuticle plays a key role in maintaining the insect's physiological function and behavior. Herein, the yellow-y protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. However, yellow-y can also have other functions, for instance, in insect behavior, but not much is known. In this study, we have studied the yellow-y gene in one important model and pest species, namely the German cockroach (Blattella germanica), which is to our knowledge the first time reported. In essence, we identified the yellow-y gene (BgY-y) and characterized its function by using RNA interference (RNAi). Silencing of BgY-y gene led to different developmental abnormalities (body weight and wings) in both genders. Specifically, there was an abundant decrease in melanin, turning the body color in pale yellow and the cuticle softer and more transparent. Interestingly, we also observed that the knockdown of BgY-y impaired the male cockroaches to display a weaker response to female-emitted contact sex pheromones, and also that the oviposition ability was weakened in the RNAi females. This study comprehensively analyzed the biological functions of the yellow-y gene in German cockroaches from the perspectives of development, body color, courtship behavior and oviposition, and as a consequence, this may opens new avenues to explore it as a novel pest control gene.
Asunto(s)
Blattellidae , Proteínas de Insectos , Oviposición , Pigmentación , Interferencia de ARN , Animales , Blattellidae/genética , Blattellidae/fisiología , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Pigmentación/genética , Cortejo , Melaninas/metabolismo , Conducta Sexual AnimalRESUMEN
Genes involved in melanin production directly impact insect pigmentation and can affect diverse physiology and behaviours. The role these genes have on sex behaviour, however, is unclear. In the present study, the crucial melanin pigment gene black was functionally characterised in an urban pest, the German cockroach, Blattella germanica. RNAi knockdown of B. germanica black (Bgblack) had no effect on survival, but did result in black pigmentation of the thoraxes, abdomens, heads, wings, legs, antennae, and cerci due to cuticular accumulation of melanin. Sex-specific variation in the pigmentation pattern was apparent, with females exhibiting darker coloration on the abdomen and thorax than males. Bgblack knockdown also resulted in wing deformation and negatively impacted the contact sex pheromone-based courtship behaviour of males. This study provides evidence for black function in multiple aspects of B. germanica biology and opens new avenues of exploration for novel pest control strategies.
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Blattellidae , Melaninas , Pigmentación , Animales , Blattellidae/genética , Blattellidae/fisiología , Masculino , Femenino , Pigmentación/genética , Melaninas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Conducta Sexual Animal , Interferencia de ARNRESUMEN
This study aimed to explore the correlation between rhizosphere soil microorganisms of wild Arnebia euchroma and the content of medicinal components to provide guidance for the selection of the ecological planting base. The total DNA of rhizosphere soil microorganisms of wild A. euchroma was extracted, and the microbial community structure of rhizosphere soil microorganisms was analyzed by IlluminaMiseq high-throughput sequencing technology. The content of total hydroxynaphthoquinone pigment and ß,ß'-dimethylacrylalkannin in medicinal materials was determined by high-performance liquid chromatography(HPLC). The physicochemical pro-perties of rhizosphere soil of wild A. euchroma in main producing areas were determined, and the correlation of soil microbial abundance with index component content and soil physicochemical properties was analyzed by SPSS software. The results showed that the species composition of rhizosphere fungi and bacteria in A. euchroma from different habitats was similar at the phylum and genus levels, but their relative abundance, richness index(Chao1), and community diversity(Simpson) index were different. Correlation analysis showed that the content of available phosphorus in soil was positively correlated with the content of total hydroxynaphthoquinone pigment and ß,ß'-dimethylacrylalkannin, and the abundance of five fungal genera such as Solicoccozyma and six bacterial genera such as Pseudo-nocardia and Bradyrhizobium was positively correlated with the content of medicinal components in medicinal materials. The abundance of Bradyrhizobium was significantly positively correlated with the content of ß,ß'-dimethylacrylalkanin. The abundance of fungi such as Archaeorhizomyces was significantly positively correlated with the content of available phosphorus in rhizosphere soil, and Bradyrhizobium was significantly negatively correlated with soil pH. Therefore, the abundance of fungi and bacteria in the rhizosphere of A. euchroma has a certain correlation with the medicinal components and the physicochemical properties of the rhizosphere soil, which can provide a scientific basis for the selection of ecological planting bases in the later stage.
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Boraginaceae , Rizosfera , Microbiología del Suelo , Bacterias/genética , Fósforo , SueloRESUMEN
Four Preyssler P5W30 based inorganic-organic hybrids, formulated as {[Cu12(pbtz)2(Hpbtz)2(OH)4(H2O)16][Na(H2O)P5W30O110]}·16H2O (1; H2pbtz = 5'-(pyridin-2-yl)-1H,2'H-3,3'-bi(1,2,4-triazole)), {[Cu10(ttbz)2(Httbz)4(OH)6(H2O)8][K(H2O)H2P5W30O110]}·30H2O (2; Httbz = 1-(tetrazo-5-yl)-4-(triazo-1-yl)benzene), {[Ni6(bpz)6(H2O)16][Na(H2O)H2P5W30O110]}·36H2O (3; bpz = 3,3',5,5'-tetramethyl-4,4-bipyrazole), {[Co4(bpz)6(H2O)9][K(H2O)H6P5W30O110]}·46H2O (4), have been isolated and structurally identified via microanalysis, thermogravimetry (TG), infrared (IR) spectroscopy, and X-ray single-crystal diffraction. Compound 1 exhibits a 3D binodal (3,6)-connected ant framework composed of dodeca-supported P5W30 polyoxometalate (POM) clusters and discrete [Cu6(pbtz)(Hpbtz)(OH)2(H2O)8] subunits. Compound 2 is a pillared-layer 3D network constructed from [Cu5(ttbz)(Httbz)2(OH)3(H2O)4] sheets pillared by individual P5W30 clusters. Compound 3 contains octa-supporting P5W30 POM clusters and novel [Ni6(bpz)6] crown-like metallamacrocycles, which construct a (4,4)-connected pts network. Compound 4 displays a complicated 3D (5,5)-connected {4(5)·6(4)·8}{4(5)·6(5)} network built by pentasupporting P5W30 POM clusters and discrete [Co4(bpz)6(H2O)9] subunits. In 1-4, the unified features are the Preyssler-type [P5W30O110] POM as the fundamental building block, which supports the transition-metal compounds with different modes to give the resultant diverse networks. The magnetism studies indicated antiferromagnetically coupled systems for the hexa- and pentanuclear Cu(II) units in 1 and 2, respectively. The electrochemical properties demonstrate that all compounds have electrocatalytic abilities toward the reduction of hydrogen peroxide. Furthermore, the catalytic activities of 1 in the cyanosilylation of aldehydes reaction have been investigated.
RESUMEN
Two extended Preyssler P5W30 polyoxometalate-based inorganic-organic hybrid materials exhibiting anion-induced supramolecular isomerism were reported. Because of the cis-trans isomerism in the octahedral CoN2O4 coordination geometry, the Preyssler P5W30 polyoxometalates are extended by double O-Co-O bridges in 1α and a single O-Co-O bridge in 1ß to form the isomeric 2D 4-connected 4(4)-sql and 3D 8-connected bcu networks, respectively. Both compounds show electrocatalytic abilities on the reduction of H2O2.
RESUMEN
Vacuolar-type (H+)-ATPase (vATPase) is a conserved multi-subunit eukaryotic enzyme composed of 14 subunits that form a functional complex consisting of an ATP-hydrolytic domain (V1) and a proton-translocation domain (V0). ATP hydrolysis and subsequent H+ translocation rely heavily on a fully assembled V1/V0 complex. Since vATPase is crucial for insect survival, it is a viable molecular target for pest control. However, detailed functional analyses of the 14 subunits and their suitability for pest control have not been fully explored in a single insect species. In this study, we identified 22 vATPase subunit transcripts that correspond to 13 subunits (A1, A2, B, C, D, E, F, G, H, a1, a2, c and d) in the white-backed planthopper (WBPH), Sogatella furcifera, a major hemipteran pest of rice. RNAi screens using microinjection and spray-based methods revealed that the SfVHA-F, SfVHA-a2 and SfVHA-c2 subunits are critical. Furthermore, star polymer (SPc) nanoparticles were utilized to conduct spray-induced and nanoparticle-delivered gene silencing (SI-NDGS) to evaluate the pest control efficacy of RNAi targeting the SfVHA-F, SfVHA-a2 and SfVHA-c2 transcripts. Target mRNA levels and vATPase enzymatic activity were both reduced. Honeydew excreta was likewise reduced in WBPH treated with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. To assess the environmental safety of the nanoparticle-wrapped dsRNAs, Cyrtorhinus lividipennis Reuter, a major natural enemy of planthoppers, was also sprayed with dsRNAs targeting SfVHA-F, SfVHA-a2 and SfVHA-c2. Post-spray effects of dsSfVHA-a2 and dsSfVHA-c2 on C. lividipennis were innocuous. This study identifies SfVHA-a2 and SfVHA-c2 as promising targets for biorational control of WBPH and lays the foundation for developing environment-friendly RNAi biopesticides.
Asunto(s)
Hemípteros , Heterópteros , Oryza , Plaguicidas , Animales , Oryza/genética , Interferencia de ARN , Medición de Riesgo , Adenosina TrifosfatoRESUMEN
RNA interference (RNAi) is a widespread post-transcriptional silencing mechanism that targets homologous mRNA sequences for specific degradation. An RNAi-based pest management strategy is target-specific and considered a sustainable biopesticide. However, the specific genes targeted and the efficiency of the delivery methods can vary widely across species. In this study, a spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system that incorporated gene-specific dsRNAs targeting conserved genes was used to evaluate phenotypic effects in white-backed planthopper (WBPH). At 2 days postspraying, transcript levels for all target genes were significantly reduced and knockdown of two gene orthologs, hsc70-3 and PP-α, resulted in an elevated mortality (>60%) and impaired ecdysis. These results highlight the utility of the SI-NDGS system for identifying genes involved in WBPH growth and development that could be potentially exploitable as high mortality target genes to develop an alternative method for WBPH control.
Asunto(s)
Genes Letales , Hemípteros , Animales , Interferencia de ARN , Silenciador del Gen , Hemípteros/genéticaRESUMEN
Centrin is a member of the EF-hand superfamily that is phosphorylated during mitosis and is associated with alterations of contractile fibers. To obtain insight into the structural basis for the functional effects of phosphorylation, we found that the serine residue at position 166 of Euplotes octocarinatus centrin (EoCen) can be phosphorylated by protein kinase A (PKA) in the absence or presence of metal ions using (31)P-NMR spectroscopy. Cations of Ca(2+) and Tb(3+) bound to EoCen resulted in an important structural transition from a closed to an open state. EoCen in both the closed and the open state can be phosphorylated by PKA. After phosphorylation, secondary and tertiary structural changes of EoCen, mainly on its C-terminal domain (C-EoCen), were noted through circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and 2-p-toluidinylnaphthalene-6-sulfonate fluorescence. After the protein was phosphorylated, the α-helix content and the extent of the exposed hydrophobic surface on EoCen were decreased. Phosphorylated EoCen has higher affinity for the peptide melittin than nonphosphorylated EoCen. In addition, binding of melittin with phosphorylated C-EoCen was enthalpy-driven.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Euplotes , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Meliteno/metabolismo , Metales/farmacología , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , TermodinámicaRESUMEN
Primary urethral carcinoma (PUC) has a low incidence, but with high aggressiveness. Most of the patients are found in late stage, with poor prognosis. At present, chemotherapy is still the main treatment for metastatic PUC, but it has limited effect. Here, we report a case of metastatic PUC with low HER2 expression that developed disease progression after multiline therapy including chemotherapy, programmed death-1 (PD-1) inhibitors and multi-targeted receptor tyrosine kinase (RTK) inhibitor. After receiving Disitamab Vedotin(a novel antibody drug conjugate, ADC) and toripalimab (a PD-1 inhibitor), the patient achieved persistent PR, and the PFS exceeded 12 months up to now. Our report indicates that, despite the patient of metastatic PUC has low expression of HER2, it is still possible to benefit from Disitamab Vedotin combined with PD-1 inhibitor, which may reverse the drug resistance of PD-1 inhibitor and chemotherapy to a certain extent. But larger sample studies are needed to determine the efficacy of this treatment strategy and its impact on survival.
Asunto(s)
Inmunoconjugados , Neoplasias Uretrales , Humanos , Neoplasias Uretrales/tratamiento farmacológico , Inhibidores de Puntos de Control InmunológicoRESUMEN
A 120-day in situ remediation of oil-contaminated soil was carried out by using highly efficient oil-degrading bacteria. The effects of bio-enhanced remediation and changes in soil physicochemical properties and enzyme activities were investigated. Combined with metagenomic sequencing and bioinformatics analysis, the strengthening mechanism was revealed. The results showed that compared with the blank control group (Ctrl), the degradation rate of total petroleum hydrocarbons in the bioremediation group (Exp-BT) was significantly increased, reaching 81.23%. During enhanced bioremediation by highly efficient oil-degrading bacteria, the pH of the soil was stable, the oxidation capacity of the system was improved, and the electrical conductivity was in the range suitable for agricultural activities. Lipase and dehydrogenase maintained high activity during repair. In addition, the analysis of the initial contaminated soil (B0), the highly efficient oil-degrading bacteria obtained from domestication (GZ), and the soil samples after bioremediation (BT) in the obtained samples showed that, at the phylum level, the total proportion of Proteobacteria and Actinobacteria increased by 17.1%. At the genus level, the abundance of Nocardioides, Achromobacter, Gordonia, and Rhodococcus increased significantly. The species and function contribution analysis of COG and KEGG proved that the above bacterial genera had important contributions to the degradation of petroleum hydrocarbons. A high abundance of petroleum hydrocarbon-related metabolic enzymes and five petroleum hydrocarbon-related degradation genes was found in the soil after remediation:alkM, tamA, rubB, ladA, and alkB. The analysis showed that the introduction of the exogenous petroleum hydrocarbon-degrading bacteria group enhanced the metabolic activity of microorganism-related enzymes and the expression of corresponding functional genes.
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Actinobacteria , Petróleo , Bacterias/genética , Proteobacteria , AgriculturaRESUMEN
Although CRISPR/Cas9 has been widely used in insect gene editing, the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with Sogatella furcifera, the white-backed planthopper (WBPH), a significant pest of Oryza sativa (rice) that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)-CRISPR, in which Cas9 and single-guide RNAs (sgRNAs) for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females. Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components. An evaluation of the offspring indicated that the highest G0 gene-edited efficacy (56.7%) occurred in females injected 2 d posteclosion, and that those mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.
RESUMEN
Centrin, an EF-hand calcium-binding protein with high homology to calmodulin (CaM), is an essential component of microtubule-organizing center (MTOC). Lanthanide (Ln) ions can improve the stability, increase the amount and enhance the orderliness of microtubules, which are components of cytoskeleton. In order to investigate the structural basis of Ln ions on enhancing orderliness of microtubules, we characterized the binding properties of Ln ions with the isolated C-terminal domain of the Euplotes centrin (C-EoCen). Results suggested that Ln ions may occupy the canonical Ca(2+) binding sites on C-EoCen with middle affinity. Near- and far-UV CD spectra of C-EoCen displayed pronounced differences before and after additing Ln ions. The asymmetry of microenvironments of Phe on C-EoCen was changed. Using 2-p-toluidinylnaphthalene-6- sulfonate (TNS) as probe, Ln ions induced C-EoCen to undergo conformational changes from closed state to open state, resulting in exposing hydrophobic patches to external environments. Ln ions have more obvious effect on the conformation of centrin than Ca(2+). The differences found in the interactions of centrin binding with Ln ions/Ca(2+) maybe provide some insights for structural basis of centrin functions in vivo.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas Cromosómicas no Histona/química , Elementos de la Serie de los Lantanoides/farmacología , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Euplotes , Interacciones Hidrofóbicas e Hidrofílicas , Elementos de la Serie de los Lantanoides/metabolismo , Ligandos , Datos de Secuencia Molecular , Fenoles , Estructura Terciaria de Proteína/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Análisis Espectral , Sulfóxidos , Xilenos/metabolismoRESUMEN
Preferential removal of phosphate from aqueous was conducted by a novel biomass-based nanocomposite (EP-N+-Zr) with encapsulated hydrous zirconium oxide, and the biopolymer EP-N+-Zr features were described. EP-N+-Zr exhibited high selective sequestration toward phosphate when humic acid or other competing anions (Cl-, SO42-, NO3-, ClO4-) coexisted at relatively high levels. Such excellent performance of EP-N+-Zr was attributed to its specific two site structures; the embedded HZO nanoparticles and quaternary ammonia groups [N+(CH2CH3)3Cl-] bonded inside the biomass-Enteromorpha prolifera, which facilitated preferable capture towards phosphate through specific affinity and nonspecific preconcentration of phosphate ions on the basis of the ion exchange, respectively. The maximum adsorption capacity of phosphate (20 °C) as calculated by Langmuir model was 88.5 mg(P)/g. Regeneration tests showed that EP-N+-Zr could be recycled at least five times without noticeable capacity losses using binary NaOH-NaCl as eluent.
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Nanopartículas , Contaminantes Químicos del Agua , Adsorción , Biopolímeros , Concentración de Iones de Hidrógeno , Cinética , Fosfatos , Contaminantes Químicos del Agua/análisis , CirconioRESUMEN
In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were involved in GTP binding.
Asunto(s)
Euplotes/enzimología , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Factores de Terminación de Péptidos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Estructura Terciaria de Proteína/genética , Alineación de SecuenciaRESUMEN
The aim of this observational study was to test whether ABO blood type was a prognostic factor for pancreatic ductal adenocarcinoma (PDAC) patients and whether other risk factors could influence pancreatic cancer patients' survival. This study included 610 patients who were diagnosed as pancreatic cancer and had undergone radical surgery. Patients' characteristics included age, gender, tumor stage, tumor grade, adenosquamous carcinoma (ASC) status, preoperative serum carbohydrate antigen 19-9 (CA19-9) levels, preoperative serum carcinoembryonic antigen (CEA) levels, ABO blood type, smoking status, and drinking status were analyzed in this study. Cox proportional hazards regression model and Kaplan-Meier method were used to evaluate the role of prognostic factors. For pancreatic cancer patients undergoing radical surgery, the overall survival was worse for ASC patients than PDAC patients (Log-rankâ=â11.315, Pâ<â.001). Compared with ASC patients (Log-rankâ<â0.001, Pâ=â.996), PDAC patients can benefit from chemotherapy (Log-rankâ=â17.665, Pâ<â.001). For PDAC patients, O blood type had better overall survival than non-O blood type (Log-rankâ=â4.153, Pâ=â.042). Moreover, the group with higher serum levels of CA19-9 had poor prognosis compared to another group with low serum CA19-9 (Log-rankâ=â4.122, Pâ=â.042). Higher CEA levels indicated poor prognosis (Log-rankâ=â13.618, Pâ<â.001). In conclusion, ASC status was associated with overall survival of pancreatic cancer patients and cannot benefit from postoperative chemotherapy. Non-O blood type was a prognostic factor for PDAC patients.
Asunto(s)
Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Sistema del Grupo Sanguíneo ABO/sangre , Adulto , Factores de Edad , Anciano , Consumo de Bebidas Alcohólicas/epidemiología , Antígeno CA-19-9/sangre , Antígeno Carcinoembrionario/sangre , Carcinoma Adenoescamoso/patología , Carcinoma Ductal Pancreático/epidemiología , Carcinoma Ductal Pancreático/cirugía , Fumar Cigarrillos/epidemiología , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Pancreáticas/epidemiología , Neoplasias Pancreáticas/cirugía , Modelos de Riesgos Proporcionales , Factores de Riesgo , Factores SexualesRESUMEN
The aim of the present study was to evaluate the potential network of arsenic trioxide (ATO) target genes in pancreatic cancer. The DrugBank, STITCH, cBioPortal, Kaplan-Meier plotter and Oncomine websites were used to analyze the association of ATO and its target genes with pancreatic cancer. Initially, 19 ATO target genes were identified, along with their associated protein-protein interaction networks and Kyoto Encyclopedia of Genes and Genomes pathways. ATO was found to be associated with multiple types of cancer, and the most common solid cancer was pancreatic cancer. A total of 6 ATO target genes (namely AKT1, CCND1, CDKN2A, IKBKB, MAPK1 and MAPK3) were found to be associated with pancreatic cancer. Next, the mutation information of the 6 ATO target genes in pancreatic cancer was collected. A total of 20 ATO interacting genes were identified, which were mainly involved in hepatitis B, prostate cancer, pathways in cancer, glioma and chronic myeloid leukemia. Finally, the genes CCND1 and MAPK1 were detected to be prognostic factors in patients with pancreatic cancer. In conclusion, bioinformatics analysis may help elucidate the molecular mechanisms underlying the involvement of ATO in pancreatic cancer, enabling more effective treatment of this disease.
RESUMEN
Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins, which often associated with the centrosomes and basal bodies. To explore the possible structural role of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu(3+). The dependence of Lu(3+) induced self-assembly of EoCen on various chemical and physical factors, including temperature, protein concentration, ionic strength and pH, was characterized using resonance light scattering (RLS). Control experiments with different metal ions suggest that Ca(2+) and Lu(3+) bindings to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu(3+) exhibits the stronger effect, however, Mg(2+) alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength demonstrate that the lutetium(III)-dependent self-assembly is closely related to the exposure of hydrophobic cavity. Control experiment on pH value with EoCen and the fragments of it, N-terminal domain of EoCen (N-EoCen), indicates that the electrostatic effect is of small tendency to be served as the main driving force in the self-assembly of EoCen. The specific oligomerization form of the protein was exhibited by cross-linking experiment.
Asunto(s)
Proteínas de Unión al Calcio/química , Euplotes/química , Lutecio/química , Proteínas Protozoarias/química , Animales , Calcio/química , Motivos EF Hand , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Naftalenosulfonatos/química , Concentración Osmolar , Estructura Terciaria de Proteína , Proteínas Protozoarias/aislamiento & purificación , Espectrometría de Fluorescencia , TemperaturaRESUMEN
CopC, a protein involved in copper resistance, is essentially constituted by two sheets forming a Greek key beta barrel motif. The aromatic ring of Trp83, sandwiched between the two beta sheets, has numerous contacts with residues in strands beta and stabilizes the protein fold. In the paper Trp83 was mutated to Leu to study the effect of this mutation on CopC by means of fluorescence spectra and UV spectra. The experiments indicate that the mutation bind Cu(2+) with a decreased formation constant of 3.95 x 10(11) M(-1) in 20 mM PB buffer at pH 7.0; mutagenesis make hydrophobic region to be exposed to an extent. Compared with the wild, thermal stability of the mutant was shown to decrease by stronger fluorescence of TNS at 80 degrees C. The important role of aromatic residue in structure is exhibited.
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Proteínas Bacterianas/metabolismo , Mutación/genética , Pseudomonas syringae/metabolismo , Triptófano/genética , Cobre/metabolismo , Proteínas Mutantes/metabolismo , Conformación Proteica , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , TermodinámicaRESUMEN
The interaction between 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and ciliate Euplotes Octocarinatus centrin (Cen) has been studied by fluorescence spectroscopy. The binding constants of TNS with Cen were measured at different temperature in the 0.01M Hepes, pH 7.4. The binding process is exothermic and involves a positive entropy change. The negative value of enthalpy predominately contributes to the negative free energy of binding between TNS and Cen. The salt (KCl) increases the association constant of TNS and Cen. These results and resonance light scattering experiment suggest that the binding force between TNS and Cen is hydrophobic. The distance (r) between TNS and tryptophan of mutant G115W, which sheds more insight into the binding of TNS to Cen, was determined as 4.85nm based on Förster non-radiative energy transfer theory.
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Proteínas de Unión al Calcio/química , Motivos EF Hand , Naftalenosulfonatos/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Modelos Moleculares , Mutación/genética , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , VolumetríaRESUMEN
Carbohydrate antigen 19-9 (CA19-9) fails to demonstrate the predictive value for early detection pancreatic ductal adenocarcinoma (PDAC). Glypican-1 (GPC1+) exosomes may serve as a noninvasive diagnostic tool to detect early stages of PDAC. Therefore, it is necessary to explore the serum GPC1 levels and determine whether serum GPC1 serves as a novel biomarker for PDAC patients. Blood samples were collected from 156 patients with PDAC, 199 non-cancer controls, and 240 patients with other cancers. Serological levels of GPC1 were examined by enzyme-linked immunosorbent assay (ELISA). Finally, a 5-year follow-up was monitored to evaluate the correlation between serum GPC1 levels and overall survival in 156 patients with PDAC. The results suggested that levels of serum GPC1 and CA19-9 were higher in PDAC patients than that of controls (P < 0.05). Serum GPC1 levels in PDAC were different from those in gallbladder carcinoma (P < 0.001), colorectal carcinoma (P < 0.001), gastric carcinoma (P < 0.001), and prostate cancer (P < 0.001), but not hepatocellular carcinoma (P = 0.395) and cholangiocarcinoma (P = 0.724). Receiver operating characteristic curve (ROC) analysis showed that serum CA19-9 was significantly better than serum GPC1 in distinguishing PDAC patients from the controls (AUC, 95% CI: 0.908, 0.868-0.947 vs 0.795, 0.749-0.841, respectively). The serum GPC1 cannot be used as a serum diagnostic biomarker for PDAC patients. The level of serum GPC1 decreased 2 days after surgery (P = 0.001), which were not different from serum GPC1 levels in healthy control (P = 0.381). The overall survival rate was shorter in patients with high levels of serum GPC1 compared to those with low levels of serum GPC1 (log-rank = 5.16, P = 0.023). Taken together, the results indicate that high levels of serum GPC1 predict poor prognosis in PDAC patients. Serum GPC1 may be a prognosis factor for PDAC patients.