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1.
Mikrochim Acta ; 186(2): 100, 2019 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-30635742

RESUMEN

A method is described for the determination of the activity of endonuclease. It based on the deaggregation of gold nanoparticles (AuNPs) aggregated by the action of poly(diallyldimethylammonium chloride) (PDDA). A single-stranded DNA (ssDNA) is released after enzymatic cleavage catalyzed by endonuclease. The released fragments bind electrostatically to PDDA and inhibit the PDDA-induced aggregation of AuNPs. This is accompanied by a color change from blue to red and a decrease in the absorption ratio (A630/A520). Under the optimal conditions, this ratio increases linearly in the 0.001 to 1 U·µL-1 EcoRI endonuclease activity range. The detection limit is of 2 × 10-4 U·µL-1 which is much better or at least comparable to previous reports. The method is deemed to have wide scope in that it may be used to study other endonuclease activity (such as BamHI) by simply changing the specific recognition site of the hairpin-like DNA probe. The assay may also be employed to screening for inhibitors of EcoRI endonuclease. Graphical abstract Schematic presentation of the colorimetric assay based on the deaggregation of AuNPs for the detection of endonuclease activity. A single-stranded sequence (ssDNA) is released by the EcoRI cleavage, which electrostatically binds to PDDA and inhibits the PDDA-induced aggregation of AuNPs accompanying with a color change from blue to red.


Asunto(s)
Colorimetría/métodos , Sondas de ADN/química , Endonucleasas/metabolismo , Oro , Secuencias Invertidas Repetidas , Nanopartículas del Metal/química , Colorimetría/normas , Desoxirribonucleasa EcoRI/antagonistas & inhibidores , Desoxirribonucleasa EcoRI/metabolismo , Endonucleasas/antagonistas & inhibidores , Límite de Detección , Polietilenos/química , Polietilenos/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo
2.
J Appl Microbiol ; 123(3): 582-593, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28419654

RESUMEN

Synthetic plastics, which are widely present in materials of everyday use, are ubiquitous and slowly-degrading polymers in environmental wastes. Of special interest are the capabilities of microorganisms to accelerate their degradation. Members of the metabolically diverse genus Pseudomonas are of particular interest due to their capabilities to degrade and metabolize synthetic plastics. Pseudomonas species isolated from environmental matrices have been identified to degrade polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyurethane, polyethylene terephthalate, polyethylene succinate, polyethylene glycol and polyvinyl alcohol at varying degrees of efficiency. Here, we present a review of the current knowledge on the factors that control the ability of Pseudomonas sp. to process these different plastic polymers and their by-products. These factors include cell surface attachment within biofilms, catalytic enzymes involved in oxidation or hydrolysis of the plastic polymer, metabolic pathways responsible for uptake and assimilation of plastic fragments and chemical factors that are advantageous or inhibitory to the biodegradation process. We also highlight future research directions required in order to harness fully the capabilities of Pseudomonas sp. in bioremediation strategies towards eliminating plastic wastes.


Asunto(s)
Plásticos/metabolismo , Pseudomonas/metabolismo , Biodegradación Ambiental , Plásticos/síntesis química , Polietilenos/metabolismo , Poliestirenos/metabolismo , Pseudomonas/clasificación , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , Succinatos/metabolismo
3.
Environ Sci Technol ; 48(23): 13776-84, 2014 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-25384056

RESUMEN

Polyethylene (PE) has been considered nonbiodegradable for decades. Although the biodegradation of PE by bacterial cultures has been occasionally described, valid evidence of PE biodegradation has remained limited in the literature. We found that waxworms, or Indian mealmoths (the larvae of Plodia interpunctella), were capable of chewing and eating PE films. Two bacterial strains capable of degrading PE were isolated from this worm's gut, Enterobacter asburiae YT1 and Bacillus sp. YP1. Over a 28-day incubation period of the two strains on PE films, viable biofilms formed, and the PE films' hydrophobicity decreased. Obvious damage, including pits and cavities (0.3-0.4 µm in depth), was observed on the surfaces of the PE films using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The formation of carbonyl groups was verified using X-ray photoelectron spectroscopy (XPS) and microattenuated total reflectance/Fourier transform infrared (micro-ATR/FTIR) imaging microscope. Suspension cultures of YT1 and YP1 (10(8) cells/mL) were able to degrade approximately 6.1 ± 0.3% and 10.7 ± 0.2% of the PE films (100 mg), respectively, over a 60-day incubation period. The molecular weights of the residual PE films were lower, and the release of 12 water-soluble daughter products was also detected. The results demonstrated the presence of PE-degrading bacteria in the guts of waxworms and provided promising evidence for the biodegradation of PE in the environment.


Asunto(s)
Bacillus/fisiología , Biopelículas/crecimiento & desarrollo , Enterobacter/fisiología , Contaminantes Ambientales/metabolismo , Mariposas Nocturnas/microbiología , Polietilenos/metabolismo , Animales , Bacillus/metabolismo , Biodegradación Ambiental , Enterobacter/metabolismo , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Larva/metabolismo , Larva/microbiología , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo
4.
J Appl Microbiol ; 116(2): 295-303, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24165295

RESUMEN

AIM: Polyethylene succinate (PES) contains hydrolysable ester bonds that make it a potential substitute for polyethylene (PE) and polypropylene (PP). Towards bioremediation of PES, we have already reported that a new strain of Pseudomonas, Pseudomonas sp. AKS2, can efficiently degrade PES and hypothesized that cell surface hydrophobicity plays an important role in this degradation process. In this study, our efforts were targeted towards establishing a correlation between cell surface hydrophobicity and PES degradation. METHODS AND RESULTS: We have manipulated cell surface hydrophobicity of AKS2 by varying concentrations of glucose and ammonium sulphate in the growth medium and subsequently examined the extent of PES degradation. We observed an increase in PES degradation by AKS2 with an increase in cell surface hydrophobicity. The increased surface hydrophobicity caused an enhanced biofilm formation on PES surface that resulted in better polymer degradation. CONCLUSION: The current study establishes a direct correlation between cell surface hydrophobicity of an organism and its potential to degrade a nonpolar polymer like PES. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell surface hydrophobicity manipulation can be used as an important strategy to increase bioremediation of nonpolar polymer like PES.


Asunto(s)
Polietilenos/metabolismo , Pseudomonas/metabolismo , Succinatos/metabolismo , Sulfato de Amonio/metabolismo , Adhesión Bacteriana , Biodegradación Ambiental , Biopelículas/crecimiento & desarrollo , Membrana Celular/metabolismo , Medios de Cultivo/química , Glucosa/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Polisacáridos Bacterianos/aislamiento & purificación , Poliestirenos/metabolismo , Pseudomonas/fisiología
5.
Biomacromolecules ; 14(8): 2570-81, 2013 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-23841591

RESUMEN

Despite the numerous vital functions of proteins in the cytosolic compartment, less attention has been paid to the delivery of protein drugs to the cytosol than to the plasma membrane. To address this issue and effectively deliver charged proteins into the cytoplasm, we used endosomolytic, thiol-triggered degradable polyelectrolytes as carriers. The cationic, reducible polyelectrolyte RPC-bPEI(0.8 kDa)2 was synthesized by the oxidative polymerization of thiolated branched polyethyleneimine (bPEI). The polymer was converted to the anionic, reducible polyelectrolyte RPA-bPEI(0.8 kDa)2 by introducing carboxylic acids. The two reducible polyelectrolytes (RPC-bPEI(0.8 kDa)2 and RPA-bPEI(0.8 kDa)2) were complexed with counter-charged model proteins (bovine serum albumin (BSA) and lysozyme (LYZ)), forming polyelectrolyte/protein complexes of less than 200 nm in size at weight ratios (WR) of ≥1. The resultant complexes maintained a proton buffering capacity nearly equivalent to that of the polyelectrolytes in the absence of protein complexation and were cytocompatible with MCF7 human breast carcinoma cells. Under cytosol-mimicking thiol-rich conditions, RPC-bPEI(0.8 kDa)2/BSA and RPA-bPEI(0.8 kDa)2/LYZ complexes increased significantly in size and released the loaded protein, unlike the protein complexes with nonreducible polyelectrolytes (bPEI(25 kDa) and bPEI(25 kDa)COOH). The polyelectrolyte/protein complexes showed cellular uptake similar to that of the corresponding proteins alone, but the former allowed more protein to escape into the cytosol from endolysosomes than the latter as a result of the endosomolytic function of the polyelectrolytes. In addition, the proteins in the polyelectrolyte/protein complexes kept their intrinsic secondary structures. In conclusion, the results show the potential of the designed endosomolytic, reducible polyelectrolytes for the delivery of proteins to the cytosol.


Asunto(s)
Portadores de Fármacos/química , Endosomas/metabolismo , Iminas/síntesis química , Nanopartículas/química , Polietilenos/síntesis química , Transporte Biológico , Tampones (Química) , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Electrólitos/química , Electrólitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Iminas/metabolismo , Células MCF-7 , Muramidasa/química , Muramidasa/metabolismo , Nanopartículas/metabolismo , Oxidación-Reducción , Tamaño de la Partícula , Polietilenos/metabolismo , Polimerizacion , Unión Proteica , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Volumetría
6.
Clin Oral Investig ; 17(3): 775-83, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22752296

RESUMEN

OBJECTIVES: To study the effectiveness of a dentifrice containing polyvinylmethyl ether-maleic acid (PVM/MA) copolymer in occluding dentin tubules and investigate the interaction between PVM/MA and type I collagen using surface plasmon resonance (SPR). MATERIALS AND METHODS: Fifteen volunteers brushed dentin discs in situ using dentifrices with and without PVM/MA copolymer in a cross-over design. Dentin tubule occlusion was evaluated after brushing, after overnight saliva challenge in vivo for 12 h and after drinking 250 ml of orange juice. Dentin tubule occlusion and tubule size were compared between the two groups using repeated ANOVA and before and after erosive challenges using paired t tests. SPR using type I collagen as ligand and PVM/MA as analyte was performed to evaluate the binding of the two macromolecules. RESULTS: A median of 91% of dentin tubules were occluded after a single brushing in the PVM/MA group, as compared to 9% in the controls. After overnight saliva challenge and 10 min of erosion by orange juice, a median of 73% of the dentin tubules remained fully occluded in the PVM/MA group as compared to zero in the controls. Dentin tubule size increased after orange juice erosion in the controls but not in the PVM/MA group. SPR study showed that PVM/MA bound readily to collagen molecules in a 4 to 1 ratio. CONCLUSIONS: Dentifrice containing PVM/MA could effectively occlude dentin tubules and prevent dentin erosion. PVM/MA may improve adhesive retention of intra-tubular dentifrice plugs through binding to dentin surface collagen. CLINICAL RELEVANCE: Brushing with dentifrice containing adhesive polymers has preventive effect against dentin erosion and dentin sensitivity.


Asunto(s)
Dentífricos/química , Dentífricos/uso terapéutico , Sensibilidad de la Dentina/prevención & control , Dentina/efectos de los fármacos , Maleatos/uso terapéutico , Polietilenos/uso terapéutico , Erosión de los Dientes/prevención & control , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Estudios Cruzados , Recubrimiento Dental Adhesivo , Dentina/ultraestructura , Método Doble Ciego , Femenino , Humanos , Masculino , Maleatos/química , Maleatos/metabolismo , Polietilenos/química , Polietilenos/metabolismo , Resonancia por Plasmón de Superficie
7.
Biomacromolecules ; 13(10): 3099-107, 2012 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-22920572

RESUMEN

Low-molecular-weight poly(ethylene carbonate) diols of varying molecular weight were generated through catalyzed thermal degradation of high-molecular-weight poly(ethylene carbonate). These polymers were then end functionalized with acrylate groups. The resulting α,ω-diacrylates were effectively photo-cross-linked upon exposure to long-wave UV light in the presence of a photoinitiator to yield rubbery networks of low sol content. The degree of cross-linking effectively controlled the in vivo degradation rate of the networks by adherent macrophages; higher cross-link densities yielded slower degradation rates. The cross-link density did not affect the number of adherent macrophages at the elastomer/tissue interface, indicating that cross-linking affected the susceptibility of the elastomer to degradative species released by the macrophages. The reactive species likely responsible for in vivo degradation appears to be superoxide anion, as the in vivo results were in agreement with in vitro degradation via superoxide anion, while cholesterol esterase, known to degrade similar poly(alkylene carbonate)s, had no affect on elastomer degradation.


Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Elastómeros/metabolismo , Macrófagos/metabolismo , Polietilenos/metabolismo , Animales , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/química , Elastómeros/síntesis química , Elastómeros/química , Macrófagos/química , Masculino , Estructura Molecular , Peso Molecular , Procesos Fotoquímicos , Polietilenos/síntesis química , Polietilenos/química , Ratas , Ratas Wistar
8.
Cells ; 11(20)2022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-36291134

RESUMEN

As a novel cell type from eight-cell-stage embryos, extended pluripotent stem cells (EPSCs) are known for diverse differentiation potency in both extraembryonic and embryonic lineages, suggesting new possibilities as a developmental research model. Although various features of EPSCs have been defined, their ability to directly transfer extended pluripotency to differentiated somatic cells by cell fusion remains to be elucidated. Here, we derived EPSCs from eight-cell mouse embryos and confirmed their extended pluripotency at the molecular level and extraembryonic differentiation ability. Then, they were fused with OG2+/- ROSA+/- neural stem cells (NSCs) by the polyethylene-glycol (PEG)-mediated method and further analyzed. The resulting fused hybrid cells exhibited pluripotential markers with upregulated EPSC-specific gene expression. Furthermore, the hybrid cells contributed to the extraembryonic and embryonic lineages in vivo and in vitro. RNA sequencing analysis confirmed that the hybrid cells showed distinct global expression patterns resembling EPSCs without parental expression of NSC markers, indicating the complete acquisition of extended pluripotency and the erasure of the somatic memory of NSCs. Furthermore, ultrastructural observation and metabolic analysis confirmed that the hybrid cells rearranged the mitochondrial morphology and bivalent metabolic profile to those of EPSCs. In conclusion, the extended pluripotency of EPSCs could be transferred to somatic cells through fusion-induced reprogramming.


Asunto(s)
Células Madre Pluripotentes , Ratones , Animales , Fusión Celular , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Polietilenos/metabolismo , Glicoles
9.
Anal Biochem ; 412(2): 183-8, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21284925

RESUMEN

Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Cromatografía de Afinidad , Polietilenos/metabolismo , Proteína Estafilocócica A/metabolismo , Animales , Línea Celular Tumoral , ADN/metabolismo , Humanos , Ratones , Células 3T3 NIH , Reacción en Cadena de la Polimerasa
10.
Microbiol Spectr ; 9(3): e0097621, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34730414

RESUMEN

Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.


Asunto(s)
Agaricales/metabolismo , Biodegradación Ambiental , Hidrolasas de Éster Carboxílico/metabolismo , Poliésteres/metabolismo , Tereftalatos Polietilenos/metabolismo , Polietilenos/metabolismo , Succinatos/metabolismo , Agaricales/enzimología , Agaricales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cacao/genética , Hidrolasas de Éster Carboxílico/genética , Contaminantes Ambientales/metabolismo , Contaminación Ambiental/análisis , Escherichia coli/genética , Escherichia coli/metabolismo , Amplificación de Genes/genética , Expresión Génica/genética , Técnicas de Amplificación de Ácido Nucleico , Plásticos/metabolismo
11.
Bioconjug Chem ; 21(9): 1602-11, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20695432

RESUMEN

A series of novel cationic polymers poly(hydroxyalkylene imines) were synthesized and tested for their ability to transfect cells in vitro and in vivo. Poly(hydroxyalkylene imines), in particular, poly(2-hydroxypropylene imine) (pHP), poly(2-hydroxypropylene imine ethylene imine) (pHPE), and poly(hydroxypropylene imine propylene imine) (pHPP) were synthesized by polycondensation reaction from 1,3-diamino-2-propanol and the appropriate dibromide. Electron microscopic examination demonstrated that the resulting polymers condensed DNA into toroid shape complexes of 100-150 nm in size. Transfection studies showed that all three polymers were able to deliver genetic material into the cell, with pHP being superior to pHPP and pHPE. pHP acted as an efficient gene delivery agent in a variety of different cell lines and outcompeted most of the widely used polymer or lipid based transfection reagents. Intravenous administration of pHP-DNA polyplexes in mice followed by the reporter gene analysis showed that the reagent was suitable for in vivo applications. In summary, the results indicate that pHP is a new efficient reagent for gene delivery in vitro and in vivo.


Asunto(s)
Alquenos/síntesis química , ADN/administración & dosificación , Iminas/síntesis química , Polímeros/síntesis química , Transfección/métodos , Alquenos/química , Alquenos/metabolismo , Animales , Aziridinas/síntesis química , Aziridinas/química , Aziridinas/metabolismo , Cationes/química , Línea Celular , ADN/química , ADN/metabolismo , Genes Reporteros , Haplorrinos , Humanos , Iminas/química , Iminas/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Tamaño de la Partícula , Polietilenos/síntesis química , Polietilenos/química , Polietilenos/metabolismo , Polímeros/química , Polímeros/metabolismo , Ratas
12.
Inflamm Res ; 59(12): 1091-7, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20607583

RESUMEN

OBJECTIVES: We have reported that oral erythromycin (EM) inhibits periprosthetic tissue inflammation in a group of patients with aseptic loosening. The purpose of this study was to assess the efficacy of local, periprosthetic EM delivery in a rat model. METHODS: Uncoated Ti pins were press-fit into the right tibia of fourteen Sprague-Dawley rats following an intramedullar injection of UHMWPE (ultra high molecular weight polyethylene) particles. Revision surgeries were performed 2 months after the primary surgery. EM was applied to the Peri-Apatite™ (PA) layer of the titanium (Ti) pins. The previously implanted Ti pins were withdrawn and replaced with Ti pins coated either with (n = 7) or without (n = 7) EM. The rats were killed 1 month after "revision surgery". The EM efficacy was evaluated by (MicroCT) µCT and histology. RESULTS: µCT analysis showed that bone volume percentage (BV/TV) was significantly higher in the EM-treated group compared to the untreated group (p < 0.05). Histological analysis showed that EM treatment inhibits UHMWPE particle-induced periprosthetic tissue inflammation compared to the untreated group. CONCLUSION: This study demonstrated that periprosthetic EM delivery reduced periprosthetic inflammation and improved the quality of surrounding bone.


Asunto(s)
Antibacterianos , Eritromicina , Implantes Experimentales , Inflamación , Osteólisis , Falla de Prótesis , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Materiales Biocompatibles Revestidos/metabolismo , Eritromicina/administración & dosificación , Eritromicina/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/patología , Osteólisis/tratamiento farmacológico , Osteólisis/etiología , Osteólisis/patología , Polietilenos/metabolismo , Ratas , Ratas Sprague-Dawley , Tibia/patología , Titanio/metabolismo
13.
Mol Ther ; 17(8): 1411-7, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19532143

RESUMEN

The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved.


Asunto(s)
ADN/metabolismo , Endocitosis/fisiología , Iminas/metabolismo , Polietilenos/metabolismo , Transfección/métodos , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Células CHO , Supervivencia Celular , Cricetinae , Cricetulus , ADN/química , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Iminas/química , Ratones , Polietilenos/química
14.
J Biomed Mater Res B Appl Biomater ; 108(5): 1993-2004, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31876393

RESUMEN

Highly cross-linked, ultrahigh molecular weight polyethylene (HXLPE) acetabular liners are inherently associated to a risk of fatigue failure due to femoral neck impingement. Different thicknesses and designs employed with HXLPE liners greatly affect mechanical loading scenario. The purpose of this study was to clarify the influence of liner offset (lateralization) and locking mechanism (presence/absence of anti-rotation tabs in the external surface) on fatigue durability in annealed and vitamin E-blended HXLPE liners with a current commercial design. Each liner tested had six anti-rotation tabs, which were engaged in the 6 of 12 recesses on the metal shell. The remaining six recesses had no direct contact with the liner, where HXLPE was mechanically unsupported by the metal backing. These mated and/or unmated rim regions in the offset (2, 3, 4-mm lateralized) liners were exposed to severe neck impingement until crack propagation was identified. Phase volume percentages (crystalline, amorphous, and intermediate phase contents) of HXLPE liners were compared before and after impingement in order to interpret differences in impingement micromechanics associated with the rim design variations. Our results showed that the presence of unmated recesses served as a stress concentrator due to the formation of millimeter-scale gaps between the liner and shell. Another potential design problem drawn from our study was liner offset associated with a small volume protruding above the metal rim. Therefore, surgeons should take special care in selecting locking designs and geometries especially when using HXLPE offset liners.


Asunto(s)
Acetábulo/cirugía , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Polietilenos/química , Polietilenos/metabolismo , Artroplastia de Reemplazo de Cadera , Reactivos de Enlaces Cruzados/química , Prótesis de Cadera , Humanos , Fenómenos Mecánicos , Metales/química , Diseño de Prótesis , Falla de Prótesis , Espectrometría Raman , Vitamina E/química
15.
Biomacromolecules ; 10(4): 798-804, 2009 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-19226174

RESUMEN

The assembly of dissolved technical lignins in aqueous and organic medium has been studied at the solid-liquid interface. Adsorption of alkali lignin onto gold coated crystals treated with a cationic polymer was determined using a quartz crystal microbalance with dissipation monitoring. Complete coverage of the cationic surface with alkali lignin occurred at low solution concentration, revealing a high affinity coefficient under both alkali and neutral conditions. With additional adsorption studies from organosolv lignin in organic solvent and spectroscopic analysis of mixtures of cationic polymer and alkali lignin, a noncovalent interaction between lignin's aromatic rings and the cation of the quaternary ammonium group was shown to exist. The work underscores how polyphenolic biopolymers can strongly interact with cations through noncovalent interactions to control molecular architecture.


Asunto(s)
Lignina/química , Lignina/metabolismo , Polietilenos/química , Polietilenos/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Adsorción , Cuarzo , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie
16.
Biomacromolecules ; 10(5): 1114-24, 2009 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-19338349

RESUMEN

We present a rapid synthetic method for the development of hyperbranched PEIs decorated with different oligosaccharide architectures as carrier systems (CS) for drugs and bioactive molecules for in vitro and in vivo experiments. Reductive amination of hyperbranched PEI with readily available oligosaccharides results in sugar functionalized PEI cores with oligosaccharide shells of different densities. These core-shell architectures were characterized by NMR spectroscopy, elemental analysis, SLS, DLS, IR, and polyelectrolyte titration experiments. ATP complexation of theses polycations was examined by isothermal titration calorimetry to evaluate the binding energy and ATP/CS complexation ratios under physiological conditions. In vitro experiments showed an enhanced cellular uptake of ATP/CS complexes compared to those of the free ATP molecules. The results arise to initiate further noncovalent complexation studies of pharmacologically relevant molecules that may lead to the development of therapeutics based on this polymeric delivery platform.


Asunto(s)
Adenosina Trifosfato/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/síntesis química , Hepatocitos/metabolismo , Iminas/metabolismo , Oligosacáridos/química , Polietilenos/metabolismo , Adenosina Trifosfato/química , Calorimetría , Humanos , Concentración de Iones de Hidrógeno , Iminas/síntesis química , Iminas/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Polietilenos/síntesis química , Polietilenos/química , Relación Estructura-Actividad , Células Tumorales Cultivadas
17.
Artículo en Inglés | MEDLINE | ID: mdl-19117810

RESUMEN

Synthetic polymeric materials such as polyethylene and polyurethane (PU) were compared to conventional adsorbents for solid phase extraction for cleaning up biological samples. Efficiency in eliminating proteins and other components usually present in biological samples, such as serum, urine, and tissues extracts, was evaluated. The assays consisted of measuring the remaining protein content in serum and tissue homogenates (liver) and collecting the spectra in the UV region for urine samples. Since the analysis of many endogenous and exogenous species in these matrices usually involves chromatographic separation, the efficiency of the clean-up procedures was also evaluated by injecting cleaned samples into a C-18 chromatographic column with UV detection. Among the investigated polymers, polytetrafluorethylene, high density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE) presented the best performance in retaining serum proteins. Proteic components of the liver homogenate were completely retained on polyurethane and polybutadiene (PB). Urine samples were cleaned by crossing columns of polytetrafluorethylene, ultra-high molecular weight polyethylene, high density polyethylene, polyurethane, and polyethylene co-butyl acrylate co-anhydride maleic (PEco), since the spectra collected after column percolation presented no peaks in the region between 190 and 390 nm. SPE cartridges showed different behavior, but along the lines of their usual performance; neither serum proteins nor urine components were retained on the phases and the liver components, though partially retained, were not desorbed with either water or methanol washes, with the exception of SAX. Chromatograms of samples cleaned with high density polyethylene showed that polymeric materials can be satisfactorily used as adsorbent for biological matrix components.


Asunto(s)
Polietilenos/química , Polímeros/química , Poliuretanos/química , Extracción en Fase Sólida/métodos , Adsorción , Animales , Proteínas Sanguíneas/metabolismo , Humanos , Hígado/metabolismo , Tamaño de la Partícula , Polietilenos/metabolismo , Polímeros/metabolismo , Poliuretanos/metabolismo , Proteinuria/metabolismo , Conejos
18.
Biochem Biophys Res Commun ; 369(3): 884-8, 2008 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-18328808

RESUMEN

It remains a mystery about the role of chondrocyte or cartilage on the co-existence of ultra-high molecular weight polyethylene (UHMWPE) wear particles from partial joint arthroplasty. An inverted co-culture system was performed to investigate the interactions between chondrocytes and UHMWPE wear particles. It was first time observed that chondrocytes can engulf UHMWPE particles and release osteoarthritis associated pro-inflammatory factors. TEM observation and flow cytometric analysis demonstrated the phagocytosis of particles by chondrocytes. It was found that polyethylene particles may reduce the viability of chondrocytes, and enhance the secretion of nitric oxide (NO) and PGE(2). In conclusion, all these phenomena may contribute to further cartilage degeneration after partial joint arthroplasty surgery. It is first identified in this study that the chondrocyte acts as phagocyte to internalize wear particles and leads to the elevations of precursor mediators of osteoarthritis.


Asunto(s)
Artroplastia de Reemplazo/efectos adversos , Cartílago Articular/inmunología , Condrocitos/inmunología , Osteoartritis/inmunología , Fagocitosis , Polietilenos/efectos adversos , Animales , Cartílago Articular/citología , Cartílago Articular/cirugía , Condrocitos/ultraestructura , Técnicas de Cocultivo , Dinoprostona/metabolismo , Citometría de Flujo , Microscopía Electrónica de Transmisión , Óxido Nítrico/metabolismo , Fagocitos/inmunología , Polietilenos/metabolismo , Sus scrofa
19.
J Microbiol Biotechnol ; 18(3): 477-82, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18388465

RESUMEN

A variety of bacterial strains were isolated from waste disposal sites of Uttaranchal, India, and some from artificially developed soil beds containing maleic anhydride, glucose, and small pieces of polyethylene. Primary screening of isolates was done based on their ability to utilize high- and low-density polyethylenes (HDPE/LDPE) as a primary carbon source. Thereafter, a consortium was developed using potential strains. Furthermore, a biodegradation assay was carried out in 500-ml flasks containing minimal broth (250 ml) and HDPE/ LDPE at 5 mg/ml concentration. After incubation for two weeks, degraded samples were recovered through filtration and subsequent evaporation. Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric-differential thermogravimetry-differential thermal analysis TG-DTG-DTA) were used to analyze these samples. Results showed that consortium-treated HDPE (considered to be more inert relative to LDPE) was degraded to a greater extent 22.41% weight loss) in comparison with LDPE (21.70% weight loss), whereas, in the case of untreated samples, weight loss was more for LDPE than HDPE (4.5% and 2.5%, respectively) at 400 degrees . Therefore, this study suggests that polyethylene could be degraded by utilizing microbial consortia in an eco-friendly manner.


Asunto(s)
Bacterias/aislamiento & purificación , Bacterias/metabolismo , Polietilenos/química , Polietilenos/metabolismo , Microbiología del Suelo , Bacterias/clasificación , Bacterias/genética , Biodegradación Ambiental , ADN Bacteriano/genética , ADN Ribosómico/genética , Análisis Diferencial Térmico , India , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiología , Espectroscopía Infrarroja por Transformada de Fourier , Termogravimetría
20.
Biointerphases ; 13(6): 06D405, 2018 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-30227717

RESUMEN

The authors report on highly swellable, stable layers of spherical dendritic glycopolymers, composed of hyperbranched poly(ethylene imine) (PEI) as core and two different maltose shells (A = dense shell and B = open shell). These glycopolymers are cross-linked and attached with poly(ethylene-alt-maleic anhydride) (PEMA) or citric acid on SiOx substrates. The swelling and adsorption of biomolecules were analyzed by spectroscopic ellipsometry and quartz crystal microbalance with dissipation. The swelling degree and complexation with the drug molecule adenosine triphosphate (ATP) were found to be up to 10 times higher for dendritic glycopolymer layers cross-linked with PEMA than for layers cross-linked with citric acid. ATP complexation by electrostatic interaction with the PEI cores was confirmed by x-ray photoelectron spectroscopy analysis. Complexation led to partial collapsing, stiffening, and increase of polymer layer viscosity of the PEMA cross-linked layers. From modeling of ellipsometric data, it was deduced that ATP complexation preferably takes place at the polymer layer-solution interface. The size effect of the adsorbates was investigated by comparing ATP complexation with the adsorption of larger vitamin B12 and human serum albumin (HSA) protein. PEI-Mal A cross-linked with PEMA was found to be resistant toward B12 and HSA adsorption due to the diffusion barrier of the polymer layer. Thus, the authors present potentially biocompatible polymer surfaces for drug loading and their surface supported release.


Asunto(s)
Adsorción , Factores Biológicos/metabolismo , Dendrímeros/metabolismo , Iminas/metabolismo , Polietilenos/metabolismo , Polisacáridos/metabolismo , Adenosina Trifosfato/metabolismo , Dendrímeros/química , Iminas/química , Polietilenos/química , Polisacáridos/química , Tecnicas de Microbalanza del Cristal de Cuarzo , Albúmina Sérica Humana/metabolismo , Análisis Espectral , Vitamina B 12/metabolismo
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