RESUMEN
Mezcal is a traditional Mexican spirit, obtained from the distillation of fermented agave juices. Its preparation has been conducted for centuries in an artisanal manner. The method used to determine the correct alcohol content is of particular interest: a stream of the liquor is poured into a small vessel to induce surface bubbles. These bubbles, known as pearls by the Mezcal artisans, remain stable for tenths of seconds only if the alcohol content is close to 50%. For higher or lower alcohol content, the bubbles burst rapidly. The long bubble lifetime is the result of surfactant-induced surface tension changes. However, the precise mechanism and its relation to alcohol content remain unexplained. In this investigation, the extended lifetime of pearls was studied both experimentally and numerically. It was found that changes in surface tension, density, viscosity (resulting from mixing ethanol and water), and the presence of surfactants are all relevant to extend the bubble lifetime. The dimensionless bubble lifetime was found to reach its maximum value when the Bond number was close to unity, corresponding to 2 mm Mezcal bubbles. These findings show that the traditional empirical method does work. Beyond this, the understanding of the process provides physical insight to many other natural and industrial problems for which the stability of surface bubbles is of importance, such as bio-foams, froth floatation, and volcanic flows.
RESUMEN
In this study, we investigate, using direct numerical simulation, the motion of a small bubble in a horizontal microchannel filled with a liquid containing surfactants. In particular, we study the combined effect of surfactants and bubble deformability on the bubble shape, bubble-liquid relative velocity, velocity field in the liquid, liquid velocity on the gas-liquid interface, and surfactant distribution on the interface. The level-set method is used to capture the gas-liquid interface. The surfactant transport equation on the gas-liquid interface is solved in an Eulerian framework and is coupled to an equation describing the transport of surfactants inside the liquid phase. The Marangoni stress, induced by surfactant concentration gradients, is computed using the continuum surface force model. The simulation results give insights into the complexity of the coupling of the different phenomena controlling the dynamics of the studied system. For instance, the results show that for values of the capillary number much smaller than unity, that is, for spherical bubbles, the bubble velocity decreases as the bubble diameter increases. Moreover, surfactants tend to decrease significantly the bubble velocity, when compared with a bubble with a clean surface. Indeed, they accumulate at a convergent stagnation point/circle on the bubble surface and deplete at a divergent stagnation point/circle. As a consequence, the velocity of the liquid adjacent to the bubble is reduced in between the convergent and divergent stagnation points/circles because of Marangoni stresses. It is shown that regarding the bubble-liquid relative velocity, the bubble behaves as a rigid sphere when the Langmuir number is larger than unity, at least for the range of parameters explored in this study. For values of the capillary number of the order of unity, the bubble can take a "bullet shape". In this case, the bubble velocity increases as the bubble diameter increases. This increase of the bubble-liquid relative velocity is linked to a drastic change in the liquid flow structure near the bubble. Surfactants are swept to the rear of the bubble and have less influence on the bubble dynamics than for spherical bubbles. Finally, it is shown that increasing the amount of surfactants adsorbing to the surface eventually leads to the bursting of the bubble.
RESUMEN
We experiment the interaction between a liquid puddle and a spherical probe by Atomic Force Microscopy (AFM) for a probe radius R ranging from 10 nm to 30 µm. We have developed a new experimental setup by coupling an AFM with a high-speed camera and an inverted optical microscope. Interaction force-distance curves (in contact mode) and frequency shift-distance curves (in frequency modulation mode) are measured for different bulk model liquids for which the probe-liquid Hamaker constant H_{pl} is known. The experimental results, analyzed in the frame of the theoretical model developed in Phys. Rev. Lett. 108, 106104 (2012)PRLTAO0031-900710.1103/PhysRevLett.108.106104 and Phys. Rev. E 85, 061602 (2012)PLEEE81539-375510.1103/PhysRevE.85.061602, allow to determine the "jump-to-contact" critical distance d_{min} below which the liquid jumps and wets the probe. Comparison between theory and experiments shows that the probe-liquid interaction at nanoscale is controlled by the liquid interface deformation. This work shows a very good agreement between the theoretical model and the experiments and paves the way to experimental studies of liquids at the nanoscale.
RESUMEN
We study the interaction between an AFM probe and a liquid film deposited over a flat substrate. We investigate the effects of the physical and geometrical parameters, with a special focus on the film thickness E, the probe radius R, and the distance D between the probe and the free surface. Deformation profiles have been calculated from the numerical simulations of the Young-Laplace equation by taking into account the probe/liquid and the liquid/substrate interactions, characterized by the Hamaker constants, Hpl and Hls. We demonstrate that the deformation of a shallow film is determined by a particular characteristic length λF = (2πγE(4)/Hls)(1/2), resulting from the balance between the capillary force (γ is the surface tension) and the van der Waals liquid/substrate attraction. For the case of a bulk liquid, the extent of the interface deformation is simply controlled by the capillary length λC = (γ/Δρg)(1/2). These trends point out two asymptotic regimes, which in turn are bounded by two characteristic film thicknesses Eg = (Hls/2πΔρg)(1/4) and Eγ = (R(2)Hls/2πγ)(1/4). For E > Eg, the bulk behavior is recovered, and for E < Eγ, we show the existence of a particular shallow film regime in which a localized tip effect is observed. This tip effect is characterized by the small magnitude of the deformation and an important restriction of its radial extent λF localized below the probe. In addition, we have found that the film thickness has a significant effect on the threshold separation distance Dmin below which the irreversible jump-to-contact process occurs: Dmin is probe radius-dependent for the bulk whereas it is film-thickness-dependent for shallow films. These results have an important impact on the optimal AFM scanning conditions.
RESUMEN
The interaction between a nanoprobe and a liquid surface is studied. The surface deformation depends on physical and geometric parameters, which are depicted by employing three dimensionless parameters: Bond number B_{o}, modified Hamaker number H_{a}, and dimensionless separation distance D. The evolution of the deformation is described by a strongly nonlinear partial differential equation, which is solved by means of numerical methods. The dynamic analysis of the liquid profile points out the existence of a critical distance D_{min}, below which the irreversible wetting process of the nanoprobe happens. For D ≥ D_{min}, the numerical results show the existence of two deformation profiles, one stable and another unstable from the energetic point of view. Different deformation length-scales, characterizing the stable liquid equilibrium interface, define the near- and the far-field deformation zones, where self-similar profiles are found. Finally, our results allow us to provide simple relationships between the parameters, which leads to determine the optimal conditions when performing atomic force microscope measurements over liquids.
Asunto(s)
Membranas Artificiales , Modelos Químicos , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Simulación por Computador , Módulo de Elasticidad , Dureza , Técnicas de Sonda Molecular , Sondas Moleculares , Propiedades de SuperficieRESUMEN
To improve transport of vaccine-loaded nanoparticles, the phage display technology was used to identify novel lead peptides targeting human M cells. Using an in vitro model of the human follicle-associated epithelium (FAE) which contains both Caco-2 and M cells, a T7 phage display library was screened for its ability either to bind the apical cell surface of or to undergo transcytosis across Caco-2 cells or FAE. The selection for transcytosis across both enterocytes and FAE identified three different peptide sequences (CTGKSC, PAVLG and LRVG) with high frequency. CTGKSC and LRVG sequences enhanced phage transport across M-like cells. When polymeric nanoparticles were grafted with the sequences CTGKSC and LRVG, their transport by FAE was significantly enhanced. These peptides could therefore be used to enhance the transport of vaccine-loaded nanoparticles across the intestinal mucosal barrier.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas , Péptidos/metabolismo , Vacunas/farmacocinética , Administración Oral , Bacteriófago T7 , Transporte Biológico , Células CACO-2 , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ligandos , Biblioteca de Péptidos , Péptidos/química , Polímeros/química , Análisis de Secuencia de ProteínaRESUMEN
Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.
Asunto(s)
Bacillus/enzimología , Variación Genética/genética , Biblioteca de Péptidos , Inhibidores de Proteasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Ácidos Alcanesulfónicos/metabolismo , Secuencia de Aminoácidos , Bacillus/genética , Sitios de Unión , Biotinilación , Catálisis , Clonación Molecular , Evolución Molecular Dirigida/métodos , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Lisina/genética , Lisina/metabolismo , Mutación/genética , Inhibidores de Proteasas/química , Procesamiento Proteico-Postraduccional , Distribución Aleatoria , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Electricidad Estática , Estreptavidina/metabolismo , Especificidad por SustratoRESUMEN
We have engineered the phage displayed TEM-1 beta-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein. Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs. Insertion points were chosen to be close enough to the active site that complex formation could affect the activity. The antibiotic resistance provided by the beta-lactamase activity was used to select the clones encoding active enzymes. Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins. Their activity could be regulated by Mab or streptavidin binding. The dissociation constants of the complexes are in the 10(-9) to 10(-6) M range. In a competitive assay, PSA could be detected at a minimal concentration of 10(-9) M. The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence. The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.
Asunto(s)
Enzimas/genética , Inmunoensayo/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , beta-Lactamasas/genética , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/metabolismo , Bacteriófagos/genética , Secuencia de Bases , Sitios de Unión , Proteínas de la Cápside , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Enzimas/inmunología , Enzimas/metabolismo , Epítopos , Datos de Secuencia Molecular , Mutación , Biblioteca de Péptidos , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Selección Genética , Estreptavidina/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , beta-Lactamasas/aislamiento & purificación , beta-Lactamasas/metabolismoRESUMEN
Transplants of cell suspensions that were either selective for granule cells or contained all hippocampal cell types were placed in the hippocampal fissure or in the infragranular cleavage plane (IGCP) of the dentate gyrus. Several transplants were found in both areas in the same dentate gyrus. After a variety of post-transplant survival times, neurons of both the donor and the host were filled with lucifer yellow in fixed sections. Sections were also immunoreacted with antibodies to glial fibrillary acidic protein (GFAP), vimentin, neural cell adhesion molecule (NCAM and HNK-1/NCAM) and were histochemically reacted for ACHE. Dendrites of neurons from transplants of cells of the whole hippocampus usually stayed within the transplant. If a dendrite from such transplants did grow out of the transplant, it grew into the molecular layer (ML) of the host dentate gyrus and not into the hilus of the host. Dendrites from granule cell selective transplants grew into the ML of the host and those that grew from fissure transplants were inverted from the normal orientation of host granule cell dendrites. Dendrites also grew out of the transplant in the absence of reactive gliosis. Transplants of cells from the whole hippocampus placed in the IGCP showed the greatest ingrowth of acetylcholinesterase (ACHE) fibers. In granule cell transplants made concurrently into the fissure and the IGCP, donor granule cell dendrites grew into the host ML from both sites, demonstrating that a gradient of tropic factors across the ML could not account for the direction and orientation of the dendritic outgrowth, since a gradient that directed the growth of one set of dendrites would work against the dendrites growing in the opposite direction.
RESUMEN
The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.
Asunto(s)
Virus Diminuto del Ratón/metabolismo , Regiones Promotoras Genéticas , Serina Endopeptidasas , Transcripción Genética , Activación Transcripcional , Proteínas no Estructurales Virales/metabolismo , Animales , Virus del Sarcoma Aviar/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Células Cultivadas , Regulación Viral de la Expresión Génica , Genes Virales , Virus del Tumor Mamario del Ratón/genética , Virus Diminuto del Ratón/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Mapeo Restrictivo , Transfección , Proteínas no Estructurales Virales/biosíntesisRESUMEN
The present experiments determined whether traumatic lesions of the dentate gyrus granule cells had a different effect on the afferents in the molecular layer (ML) than nontraumatic lesions. Nontraumatic lesions of the granule cells induced by colchicine, ibotenic acid, x-radiation, and adrenalectomy have been reported to reduce both the acetylcholinesterase (AChE)-positive fibers and entorhinal afferents in the ML. After the nontraumatic granule cell lesions, the laminar distribution of the entorhinal afferents was maintained in the ML, whereas the AChE laminar pattern was lost. In the present study, dentate granule cells were traumatically lesioned by a fluid injection into the infragranular cleavage plane (IGCP) of the dentate gyrus. The traumatic lesion resulted in an altered distribution of the afferents in the ML. The perforant path fibers, shown by injection of wheat germ agglutinin horseradish peroxidase into the entorhinal cortex, occupied a greater proportion of the ML in lesioned animals than in control animals. The normal laminar pattern of AChE-positive afferents was not present after the granule cell lesion. There was an initial increase in AChE-positive fibers in the ML that lasted several weeks but eventually returned to near normal levels. The altered distribution of afferents could in part be due to uneven shrinkage of the molecular layer and/or sprouting of the afferents. Granule cell suspension transplants into the IGCP also traumatically lesioned the host granule cells but immediately replaced the damaged host granule cells with immature granule cells. The distribution of afferents was similar to that found in lesioned-only animals. The traumatic lesion induced MAP2 immunoreactivity in the anisomorphic reactive astrocytes of the ML. At the longer survival times, MAP2 was not seen in either the astrocytes of the ML or in the isomorphic reactive astrocytes in CA3.
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Hipocampo/citología , Neuronas Aferentes/patología , Acetilcolinesterasa/metabolismo , Animales , Trasplante de Células , Hipocampo/lesiones , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Neuronas Aferentes/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Immature cells transplanted into an adult host must adapt to their new environment. In the present study we have shown the dendritic development of dentate granule cells following transplantation. The adult host granule cells were lesioned by a fluid injection into the infragranular cleavage plane of the dentate gyrus. Few, if any, granule cells survived the lesion and the molecular layer (ML) shrank. When allogeneic neonatal granule cells were included in the fluid, the host granule cells were simultaneously killed and replaced. In order to visualize the dendrites, the granule cells were filled with Lucifer yellow (LY) in fixed sections and subsequently immunoreacted with an antibody to LY. The granule cell dendrites in the transplant were shorter in length, had a greater cross-sectional area, had more spines, and were more coiled and bent than control granule cell dendrites. The dendrites in the transplant formed functional synapses as indicated by cytochrome oxidase histochemistry and the transplant prevented xc03some of the ML shrinkage. Acetylcholinesterase (ACHE) xkreaction product increased both in lesioned and in transplant groups. The laminar pattern of ACHE in the control ML was not seen after the lesion and did not return in animals with successful transplants. We conclude that (i) the dendrites of neurons in the transplant adapted to the adult host environment and a shrinking ML with remarkable structural plasticity; (ii) the transplant prevented some of the shrinkage of the ML; (iii) the transplant could not reverse some of the lesion-induced changes in host organization, such as the organization of ACHE inputs to the ML; and (iv) a phenotypically specific population of transplanted neurons can replace traumatically lesioned neurons of the same type even if the host conditions continue to change.
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Trasplante de Tejido Encefálico , Hipocampo/trasplante , Hipocampo/ultraestructura , Acetilcolinesterasa/metabolismo , Animales , Diferenciación Celular , Dendritas/ultraestructura , Hipocampo/enzimología , Masculino , Plasticidad Neuronal , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
The nonstructural (NS) transcription unit of minute virus of mice (MVMp) encodes proteins that are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, it has been shown that NS-protein accumulation is toxic for transformed cells. With the aim of identifying the NS-protein function(s) responsible for cytotoxicity, point mutations and deletions were introduced in the NS-protein-coding sequence of MVMp. This strategy indicated that in transformed human NBE cells, the NS-1 protein is indispensable for MVMp DNA replication, trans activation of the late parvoviral promoter P38, trans inhibition of the long terminal repeat promoter of the Rous sarcoma virus, and cytotoxicity. Moreover, some mutations led to the dissociation of the replicative and regulatory functions of the NS-1 protein and showed that cytotoxicity correlated with the latter, more particularly with the capacity to trans inhibit the heterologous promoter. The NS-1 sequences required for cytotoxicity were found to be restricted to the amino- and carboxy-terminal portions of the protein. Although the cytotoxicities of NS-1 extremities were weak when the extremities were tested separately, the cytotoxicities were comparable to that of the full protein when the extremities were fused. Interestingly, an overall negative charge can be predicted from the NS-1 sequence over about 100 amino acids at both ends. The conservation of this charge distribution among the NS proteins of different parvoviruses suggests that NS-1 may bear some similarities to acidic transcriptional activators.
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Cápside/genética , Virus Diminuto del Ratón/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Proteínas del Núcleo Viral/genética , Animales , Supervivencia Celular , Replicación del ADN , ADN Viral/biosíntesis , Farmacorresistencia Microbiana/genética , Electroforesis en Gel de Agar , Gentamicinas/farmacología , Ratones , Mutación , Plásmidos , Activación Transcripcional , Transformación Genética , Proteínas no Estructurales ViralesRESUMEN
The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.
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Cápside/fisiología , Transformación Celular Viral , Virus Diminuto del Ratón/patogenicidad , Parvoviridae/patogenicidad , Proteínas del Núcleo Viral/fisiología , Marcadores Genéticos , Humanos , Virus Diminuto del Ratón/genética , Regiones Promotoras Genéticas , Transcripción Genética , Transfección , Proteínas no Estructurales ViralesRESUMEN
An experiment was performed on 29 dairy goats at the onset of lactation; energy and protein deficits were maximal during the first week of lactation. This was confirmed for energy by the plasma contents on non-esterified fatty acids (NEFA), beta-OH-butyrate and glucose. However, protein deficit was more critical during the second week, as shown by plasma urea, glycine and 3-CH3-histidine contents. At that time, nutritional status depended more on udder production than on intake level. Moreover, the nutritional parameters of body lipid and protein mobilization were positively correlated. Milk protein content was positively correlated with the nutritive balance and status of the goats.