RESUMEN
Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Lisosomas/metabolismo , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Difusión/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Sacarosa/metabolismo , Sacarosa/farmacologíaRESUMEN
Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (Pseudomonas aeruginosa and Escherichia coli) growth, but retained the inhibitory activity towards biofilm formation of bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P. aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study suggests that structural variation of brominated furanones can be designed for targeted functions to control biofilm formation.
Asunto(s)
Biopelículas/efectos de los fármacos , Compuestos Bicíclicos con Puentes/química , Furanos/química , Furanos/farmacología , Pseudomonas aeruginosa/fisiología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Furanos/síntesis química , Halogenación , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacosRESUMEN
Single-chain surfactants usually emulsify and stabilize oily substances into droplets in an aqueous solution. Here, we report a coassembly system, in which single types of anionic or non-ionic surfactants emulsify a class of water-soluble nonamphiphilic organic salts with fused aromatic rings in aqueous solutions. The nonamphiphilic organic salts are in turn promoted to form droplets of water-based liquid crystals (chromonic liquid crystals) encapsulated by single-chain surfactants. The droplets, stabilized against coalescence by encapsulated in a layer (or layers) of single chain surfactants, are of both nonspherical tactoid (elongated ellipsoid with pointy ends) and spherical shapes. The tactoids have an average long axis of â¼9 µm and a short axis of â¼3.5 µm with the liquid crystal aligning parallel to the droplet surface. The spherical droplets are 5-10 µm in diameter and have the liquid crystal aligning perpendicular to the droplet surface and a point defect in the center. Cationic and zwitterionic surfactants studied in this work did not promote the organic salt to form droplets. These results illustrate the complex interplay of self-association and thermodynamic incompatibility of molecules in water, which can cause new assembly behavior, including potential formation of vesicles or other assemblies, from surfactants that usually form only micelles. These unprecedented tactoidal shaped droplets also provide potential for the fabrication of new soft organic microcapsules.
Asunto(s)
Cristales Líquidos/química , Tensoactivos/química , Emulsiones/química , Estructura Molecular , Tamaño de la Partícula , Solubilidad , Soluciones , Propiedades de Superficie , Agua/químicaRESUMEN
This work reports the synthesis of a complex of a carboplatin analog having tethered adamantane that is encapsulated in the hydrophobic cavity of ß-cyclodextrin (ßCD) and its cytotoxic activity towards human neuroblastoma cells (SK-N-SH). We found that this inclusion complex of ßCD adamantane carboplatin analog exhibited higher cytotoxicity towards SK-N-SH cells than carboplatin itself, and the inclusion complex exhibited a higher binding to plasmid pBR322 deoxyribonucleic acid (DNA) than carboplatin. Confocal fluorescence images of SK-N-SH cells treated with ßCD having an attached fluorescein isothiocyanate (FITC)-tag exhibited fluorescence in the vicinity of the nuclei of the neuroblastoma cells. Direct measurements of the platinum content in SK-N-SH cells using inductively coupled plasma mass spectrometry (ICP-MS) indicated that the uptake rate of carboplatin was about 4 times higher than ßCD adamantane carboplatin analog inclusion complex. When compared to carboplatin, we believe that the higher cytotoxicity of inclusion complex towards SK-N-SH cells is due to its higher DNA binding ability as compared to carboplatin, and more efficient delivery to the nucleus of the cell. This work suggests that the advantage of deliberate noncovalent modification with ßCD through host-guest chemistry may also be broadly applicable to other anticancer agents as well.
Asunto(s)
Adamantano/química , Antineoplásicos/química , Carboplatino/análogos & derivados , Platino (Metal)/química , beta-Ciclodextrinas/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Carboplatino/síntesis química , Carboplatino/farmacología , Carboplatino/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Fluoresceína-5-Isotiocianato/química , Humanos , Espectrometría de Masas , Microscopía Confocal , Plásmidos/metabolismoRESUMEN
This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with ß-cyclodextrins (ßCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).
Asunto(s)
Ciclodextrinas/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Multimerización de Proteína , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Temperatura , Adsorción , Animales , Lisina , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Propiedades de Superficie , Agua/químicaRESUMEN
This work describes the different durations of surface confinement of adhered mammalian cells by monolayers comprised of enantiomers of bio-inert polyol-terminated alkanethiols. Enhanced resistance to protein adsorption and cell adhesion is obtained on monolayers formed by a racemic mixture of the enantiomeric alkanethiols.
Asunto(s)
Incrustaciones Biológicas , Amidas/química , Animales , Bovinos , Adhesión Celular , Línea Celular , Manosa/química , Ratones , Oligopéptidos/química , Albúmina Sérica/química , Estereoisomerismo , Sulfuros/química , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
This work reports the resistance to protein adsorption and bacterial biofilm formation by chiral monolayers of polyol-terminated alkanethiols surrounding micrometer-sized patterns of methyl-terminated alkanethiols on gold films. We discover that patterned surfaces surrounded by chiral polyol monolayers can distinguish different stages of biofilm formation. After inoculation on the surfaces, bacteria first reversibly attached on the chiral polyol monolayers. Over time, the bacteria detached from the polyol surfaces, and attached on the hydrophobic micropatterns to form biofilms. Interestingly, while both enantiomers of gulitol- and mannonamide-terminated monolayer resisted adsorption of proteins (bovine serum albumin, lysozyme, and fibrinogen) and confined biofilms formed on the micropatterns, the monolayers formed by the racemic mixture of either pair of enantiomers exhibited stronger antifouling chemistry against both protein adsorption and biofilm formation than monolayers formed by one enantiomer alone. These results reveal the different chemistries that separate the different stages of biofilm formation, and the stereochemical influence on resisting biofoulings at a molecular-level.
Asunto(s)
Biopelículas/efectos de los fármacos , Incrustaciones Biológicas/prevención & control , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Adsorción/efectos de los fármacos , Animales , Bovinos , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Oro/química , Polímeros/química , Albúmina Sérica Bovina/química , Estereoisomerismo , Alcoholes del Azúcar/química , Compuestos de Sulfhidrilo/síntesis química , Propiedades de SuperficieRESUMEN
This work studies the phase separations between polymers and a small molecule in a common aqueous solution that do not have well-defined hydrophobic-hydrophilic separation. In addition to poly(acrylamide) (PAAm) and poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP) also promotes liquid crystal (LC) droplet formation by disodium cromoglycate (5'DSCG) solvated in water. In the presence of these polymers, the concentration of 5'DSCG needed for forming LC droplets is substantially lower than that needed for forming an LC phase by 5'DSCG alone. To define the concentration ranges that 5'DSCG molecules form liquid crystals (either as droplets or as an isotropic-LC mixture), we constructed ternary phase diagrams for 5'DSCG, water, and a polymer - PVA, PVP, or PAAm. We discovered that PAAm with high molecular weight promotes LC droplet formation by 5'DSCG more effectively than PAAm with low molecular weight. At the same weight percentage, long-chain PAAm can cause 5'DSCG to form LC droplets in water, whereas short-chain PAAm does not. Poly(vinyl pyrrolidone) (PVP), which has functional groups that are more dissimilar to 5'DSCG than PVA and PAAm, promotes LC droplet formation by 5'DSCG more effectively than either of the other two polymers. Additionally, small angle neutron scattering data revealed that the assembly structure of 5'DSCG promoted by the presence of PVA is similar to the thread structure formed by 5'DSCG alone. Together, these results reveal how noncovalent polymerization can be promoted by mixing thermodynamically incompatible molecules and elucidate the basic knowledge of nonamphiphilic colloidal science.
Asunto(s)
Polímeros/química , Agua/química , Cromolin Sódico/química , Cristalización , Interacciones Hidrofóbicas e Hidrofílicas , Cristales Líquidos/química , Estructura Molecular , Dispersión del Ángulo Pequeño , Soluciones , TermodinámicaRESUMEN
Immobilizing peptides or proteins on bioinert surfaces enables the elucidation of ligand-receptor interaction in complex biological systems. Here, we report a highly chemoselective surface reaction that immobilizes peptides exclusively via N-terminus cysteine residue in a peptide. At pH 5.5, only N-terminus cysteines of peptides couple covalently with phenoxy amino squarate moieties presented on self-assembled monolayers (SAMs) of alkanethiols on gold films. The selectivity of this surface reaction can tolerate the presence of internal cysteines in close proximity to basic residues such as histidines. We demonstrated this selective surface reaction by mammalian cell adhesion and by SAMDI mass spectroscopy of the SAMs.