RESUMO
A set of arylazopyrazole-based inhibitors targeting the mitotic motor protein CENP-E was discovered through the chemical platform using the quantitative cyclization of 1,3-diketone intermediate with various hydrazines under mild conditions. Through this efficient platform, the structure-activity relationship pertaining to the pyrazole photoswitch in photoswitchable CENP-E inhibitors not only in vitro but also in cells was successfully clarified.
Assuntos
Proteínas Cromossômicas não Histona , Pirazóis , Ciclização , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Relação Estrutura-Atividade , Humanos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Estrutura Molecular , Compostos Azo/química , Compostos Azo/farmacologia , Compostos Azo/síntese química , Relação Dose-Resposta a DrogaRESUMO
An arylazopyrazole-based covalent inhibitor targeting the mitotic motor protein of centromere-associated protein E (CENP-E) was developed. Using this photoswitchable inhibitor, a photoswitchable CENP-E was chemically constructed in cells, which enabled to local control of mitotic cell division with light illumination.
Assuntos
Proteínas Cromossômicas não Histona , Humanos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Processos Fotoquímicos , Luz , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Células HeLa , Estrutura Molecular , Mitose/efeitos dos fármacosRESUMO
The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer's disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells' Raman profiles were then discussed in view of key factors including the formation of amyloid ß (Aß) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies.
Assuntos
Doença de Alzheimer , Neuroblastoma , Humanos , Porphyromonas gingivalis , Peptídeos beta-Amiloides , Lipopolissacarídeos , Corpos de Inclusão , VesículaRESUMO
Freezing impinged water droplets on glass surfaces cause serious problems such as reduced visibility of traffic lights and surveillance cameras. Droplets in the air associated with these issues are often at subzero temperatures. However, experimental results on the freezing of precooled impinged droplets are limited. In this study, we measured the freezing of precooled and impinged water droplets on cold glass surfaces. Two types of lattice-patterned microscale grooves were formed on glass surfaces to reduce the contact area of droplets and growth of frosts, which contributed to droplet freezing. In addition, the surfaces were coated with a silane coupling agent to further reduce the contact area. We analyzed the images of droplets captured using a high-speed video camera. The results of the linear relationships between the frozen droplet height, freezing front velocity, and freezing time (for the impinged droplets) indicated that the grooves and coating were effective in retarding the freezing of impinged droplets. This retardation was more evident for frost-free glass surfaces, and it was less evident for precooled droplets. Moreover, a simple heat transfer analysis was conducted to effectively estimate the overall heat flux and freezing front velocity. The sublimation of frost (adjacent to the impinged droplets) and supercool elimination of the precooled droplets significantly contributed to the heat flux and caused an increase in the freezing front velocity.
RESUMO
We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.
Assuntos
Bacteriocinas/genética , Virgibacillus/classificação , Virgibacillus/genética , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Microbiologia Ambiental , Humanos , RNA Ribossômico 16S , Água do Mar/microbiologia , Análise de Sequência de DNA , Virgibacillus/metabolismo , Sequenciamento Completo do GenomaRESUMO
Self-assembled peptide nanofibers (NFs) obtained from ß-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of ß-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure-area (π-A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of ß-sheet peptide NFs, which may broaden its potential.
Assuntos
Membranas Artificiais , Nanofibras/química , Etilenoglicol/químicaRESUMO
Recently, nanofibers (NFs) formed from antigenic peptides conjugated to ß-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of ß-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos/farmacologia , Células Dendríticas/efeitos dos fármacos , Ovalbumina/farmacologia , Peptídeos/farmacologia , Adjuvantes Imunológicos/química , Sequência de Aminoácidos , Animais , Antígenos/química , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Etilenoglicol/química , Etilenoglicol/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Camundongos Endogâmicos C57BL , Nanofibras/química , Nanofibras/ultraestrutura , Ovalbumina/química , Peptídeos/química , Conformação Proteica em Folha betaRESUMO
The pentacyclic triterpenoid ursolic acid was previously shown to inhibit the intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1) from the endoplasmic reticulum (ER) to the Golgi apparatus. In the present study, we further investigated the biological activities of three pentacyclic triterpenoids closely related to ursolic acid on the interleukin 1α-induced expression and intracellular trafficking of ICAM-1. In human lung adenocarcinoma A549 cells, asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface. Endoglycosidase H-sensitive glycans were linked to ICAM-1 in asiatic acid-, corosolic acid-, and maslinic acid-treated cells. Unlike corosolic acid, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein. Moreover, asiatic acid increased the co-localization of ICAM-1 with calnexin (an ER marker), but not GM130 (a cis-Golgi marker). Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. These results indicate that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular transport of ICAM-1 to the cell surface and cause the accumulation of ICAM-1 linked to endoglycosidase H-sensitive glycans.
Assuntos
Retículo Endoplasmático/metabolismo , Glicosilação/efeitos dos fármacos , Complexo de Golgi/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Triterpenos Pentacíclicos/farmacologia , Triterpenos/farmacologia , Células A549 , Citocinas/imunologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/imunologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Microscopia Confocal , Polissacarídeos/metabolismo , Transporte ProteicoRESUMO
Development of protein delivery systems is important for biomedical applications such as immunotherapy. Ovalbumin (OVA) is a major component of egg whites, and is a possible cause of egg allergy. In this study, OVA was used as a model protein to develop a delivery system using guanidine-terminated dendrimers (Gdn-den) bearing an amyloid-promoting peptide derived from the helix B (hB) region of OVA (hB-Gdn-den). OVA nanoparticles (NPs) were prepared by heat treatment of OVA/hB-Gdn-den mixtures. The NP size and the surface charge were controlled by adjusting the ratio of hB-Gdn-den to OVA. The NPs were around 200 nm in diameter and stably dispersed, and their encapsulation efficiency for OVA was more than 80%. Although OVA NPs were also prepared using Gdn-den, the NPs aggregated readily. Complexation with hB-Gdn-den induced conformational changes in the OVA, and the hB peptide promoted digestion of OVA. These suggest that the hB peptide of the Gdn-den works as a possible anchor to OVA. The positively charged OVA NPs effectively associated with RAW264 cells. Thus, the amyloid-promoting Gdn-den, when mixed with OVA at a suitable molar ratio to form NPs, could act as a carrier for delivery of antigen proteins to immune cells.
Assuntos
Amiloide/química , Dendrímeros/química , Sistemas de Liberação de Medicamentos , Guanidina/química , Nanopartículas/química , Ovalbumina/administração & dosagem , Fragmentos de Peptídeos/química , Animais , Células Cultivadas , Química Farmacêutica , Macrófagos , Camundongos , Ovalbumina/metabolismoRESUMO
Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca(2+) ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca(2+) changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 °C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca(2+) activation, and stabilization.
Assuntos
Actinobacteria/enzimologia , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Ativadores de Enzimas/metabolismo , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Actinobacteria/genética , Dicroísmo Circular , Clonagem Molecular , Análise Mutacional de DNA , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrolases/química , Hidrolases/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.
Assuntos
Benzo(a)pireno , Corantes Fluorescentes , Mutação Puntual , RNA , Benzo(a)pireno/análise , Benzo(a)pireno/química , RNA/genética , RNA/química , RNA/análise , Corantes Fluorescentes/química , Colorimetria , Espectrometria de Fluorescência , Oligonucleotídeos/químicaRESUMO
We developed a centromere-associated protein E (CENP-E) inhibitor employing trans to cis photoisomerization with 405 nm visible light illumination and fast thermal relaxation. This photoswitching characteristic of the inhibitor enabled selective blockage or release of the motion of particular chromosomes within a single mitotic cell. Using this technique, we successfully demonstrated targeted chromosome gain and loss in daughter cells by introducing asymmetric chromosome segregation.
Assuntos
Proteínas Cromossômicas não Histona , Luz , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Humanos , Processos Fotoquímicos , Células HeLa , Compostos Azo/química , Compostos Azo/farmacologia , Estrutura Molecular , Segregação de Cromossomos/efeitos dos fármacosRESUMO
BACKGROUND: Cervical cancer is a major global health concern with a high prevalence in low- and middle-income countries. Natural products, particularly plant-derived compounds, have shown immense potential for developing anticancer drugs. In this study, we aimed to investigate the anticancer properties of the pericarp and seeds of Sphaerocoryne affinis fruit on human cervical carcinoma cells (HeLa) and isolate the bioactive compound from the active fraction. METHODS: We prepared solvent fractions from the ethanol extracts of the pericarp and the seed portion by partitioning and assessing their cytotoxicity on HeLa cells. Subsequently, we collected acetylmelodorinol (AM), an anticancer compound, from the ethyl acetate fraction of seeds and determined its structure using nuclear magnetic resonance. We employed cytotoxicity assay, western blotting, Annexin V apoptosis assay, measurement of intracellular reactive oxygen species (ROS) levels, 4',6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, to evaluate the anticancer properties of AM on HeLa. RESULTS: The solvent fractions from the seed displayed considerably higher cytotoxic activity against HeLa cells than those of the pericarp. We isolated and identified acetylmelodorinol as an anticancer compound from the ethyl acetate fraction from S. affinis seed extract. Treatment with acetylmelodorinol inhibited HeLa cell proliferation with an IC50 value of 2.62 ± 0.57 µg/mL. Furthermore, this study demonstrated that acetylmelodorinol treatment disrupted cell cycle progression by reducing the expression of cyclin E, CDK1/2, and AKT/mTOR pathways, increasing the intracellular ROS levels, reducing BCL-2/BCL-XL expression, causing DNA fragmentation and nuclear shrinkage, and triggering apoptosis through caspase 3 and 9 activation in a dose-and time-dependent manner. CONCLUSION: In contrast to previous reports, this study focuses on the inhibitory effects of AM on the AKT/mTOR pathway, leading to a reduction in cell proliferation in cervical cancer cells. Our findings highlight the promising potential of acetylmelodorinol as an effective treatment for cervical cancer. Additionally, this study establishes a foundation for investigating the molecular mechanisms underlying AM's properties, fostering further exploration into plant-based cancer therapies.
Assuntos
Acetatos , Proteínas Proto-Oncogênicas c-akt , Neoplasias do Colo do Útero , Feminino , Humanos , Células HeLa , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Apoptose , Proliferação de Células , Serina-Treonina Quinases TOR , Sementes , Solventes/farmacologia , Solventes/uso terapêuticoRESUMO
The amyloid fibril of a fragment of the substrate binding site of αA-crystallin (αAC(71-88)) exhibited chaperone-like activity by suppressing the aggregation of alcohol dehydrogenase (ADH) and luciferase. By contrast, the amyloid fibril of the cytotoxic fragment of amyloid ß protein (Aß(25-35)) facilitated the aggregation of the same proteins. We have determined the zeta potential of the amyloid fibril by measuring their electrophoretic mobility to study the effects of the surface charge on the modulation of protein aggregation. The αAC(71-88) amyloid possesses a large negative zeta potential value which is unaffected by the binding of the negatively charged ADH, indicating that the αAC(71-88) amyloid is stable as a colloidal dispersion. By contrast, the Aß(25-35) amyloid possesses a low zeta potential value, which was significantly reduced with the binding of the negatively charged ADH. The canceling of the surface charge of the amyloid fibril upon substrate binding reduces colloidal stability and thereby facilitates protein aggregation. These results indicate that one of the key factors determining whether amyloid fibrils display chaperone-like or antichaperone activity is their electrostatic interaction with the substrate. The surface of the αAC(71-88) amyloid comprises a hydrophobic environment, and the chaperone-like activity of the αAC(71-88) amyloid is best explained by the reversible substrate binding driven by hydrophobic interactions. On the basis of these findings, we designed variants of amyloid fibrils of αAC(71-88) that prevent protein aggregation associated with neurodegenerative disorders.
Assuntos
Amiloide/química , Amiloide/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Cadeia A de alfa-Cristalina/química , Sequência de Aminoácidos , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Animais , Humanos , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Eletricidade Estática , Proteínas tau/químicaRESUMO
Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly ß-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to ß-strands, thereby forming sequential intermolecular linkages.
Assuntos
Amiloide/química , Ovalbumina/química , Peptídeos/química , Amiloide/genética , Animais , Galinhas , Cisteína/química , Cisteína/genética , Dissulfetos/química , Mutagênese Sítio-Dirigida , Ovalbumina/genética , Peptídeos/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Curcumina , Camundongos , Animais , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Curcumina/farmacologia , Curcumina/uso terapêutico , Antioxidantes/uso terapêutico , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/uso terapêutico , Camundongos Transgênicos , Superóxido Dismutase/genética , Modelos Animais de Doenças , Medula Espinal/metabolismoRESUMO
Nucleic acids in body fluids, such as circulating cell-free nucleic acids, viral DNA, and RNA have received much attention for their great potential as biomarkers in liquid biopsies of serious diseases. Although quantitative polymerase chain reaction (qPCR) has been traditionally used as a laboratory-based assay for measuring nucleic acids, there is a strong demand for techniques to qualitatively, rapidly, and simply measure the extremely low-abundance nucleic acids in order to realize the nucleic acid-based liquid biopsies. With this aim in mind, we developed a simple and highly sensitive sandwich-type assay for nucleic acids using a combination of surface-enhanced Raman scattering (SERS), which enhances Raman scattering by 108- to 1010-fold, and bioorthogonal Raman tags, which generate signals in the biologically silent region (1800-2800 cm-1). Using gold nanorods having approximately 240 strands of oligonucleotides and 4-cyano-N-(2-mercaptoethyl)benzamide (4CMB) as the bioorthogonal Raman tag, we successfully detected target nucleic acids in a sequence-selective manner.
Assuntos
DNA/análise , Análise Espectral Raman/métodos , DNA/química , Ouro/química , Microesferas , Nanotubos/química , Hibridização de Ácido Nucleico , Sefarose/química , Propriedades de SuperfícieRESUMO
The development of anti-icing, anti-frosting transparent plates is important for many reasons, such as poor visibility through the ice-covered windshields of vehicles. We have fabricated new glass surfaces coated with polypeptides which mimic a part of winter flounder antifreeze protein. We adopted glutaraldehyde and polyethylene glycol as linkers between these polypeptides and silane coupling agents applied to the glass surfaces. We have measured the contact angle, the temperature of water droplets on the cooling surfaces, and the frost weight. In addition, we have conducted surface roughness observation and surface elemental analysis. It was found that peaks in the height profile, obtained with the atomic force microscope for the polypeptide-coated surface with polyethylene glycol, were much higher than those for the surface without the polypeptide. This shows the adhesion of many polypeptide aggregates to the polyethylene glycol locally. The average supercooling temperature of the droplet for the polypeptide-coated surface with the polyethylene glycol was lower than for the polypeptide-coated surface with glutaraldehyde and the polyethylene-glycol-coated surface without the polypeptide. In addition, the average weight of frost cover on the specimen was lowest for the polypeptide-coated surface with the polyethylene glycol. These results argue for the effects of combined polyethylene glycol and polypeptide aggregates on the locations of ice nuclei and condensation droplets. Thus, this polypeptide-coating with the polyethylene glycol is a potential contender to improve the anti-icing and anti-frosting of glasses.
Assuntos
Proteínas Anticongelantes/química , Vidro/química , Polietilenoglicóis/química , Congelamento , Gelo/análise , Microscopia de Força Atômica/métodos , Peptídeos , Propriedades de Superfície/efeitos dos fármacos , Temperatura , ÁguaRESUMO
Alzheimer's disease (AD) is proposed to be induced by abnormal aggregation of amyloidß in the brain. Here, we designed a brain-permeable peptide nanofiber drug from a fragment of heat shock protein to suppress aggregation of the pathogenic proteins. To facilitate delivery of the nanofiber into the brain, a protein transduction domain from Drosophila Antennapedia was incorporated into the peptide sequence. The resulting nanofiber efficiently suppressed the cytotoxicity of amyloid ßby trapping amyloid ß onto its hydrophobic nanofiber surface. Moreover, the intravenously or intranasally injected nanofiber was delivered into the mouse brain, and improved the cognitive function of an Alzheimer transgenic mouse model. These results demonstrate the potential therapeutic utility of nanofibers for the treatment of AD.
Assuntos
Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/administração & dosagem , Encéfalo/metabolismo , Modelos Animais de Doenças , Transtornos da Memória/prevenção & controle , Nanofibras/administração & dosagem , Placa Amiloide/prevenção & controle , Administração Intranasal , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Animais , Encéfalo/efeitos dos fármacos , Feminino , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Camundongos , Camundongos Transgênicos , Nanofibras/química , Placa Amiloide/etiologia , Placa Amiloide/patologiaRESUMO
The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates.