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The natural reservoir of Ebola virus (EBOV), agent of a zoonosis burdening several African countries, remains unidentified, albeit evidence points towards bats. In contrast, the ecology of the related Marburg virus is much better understood; with experimental infections of bats being instrumental for understanding reservoir-pathogen interactions. Experiments have focused on elucidating reservoir competence, infection kinetics and specifically horizontal transmission, although, vertical transmission plays a key role in many viral enzootic cycles. Herein, we investigate the permissiveness of Angolan free-tailed bats (AFBs), known to harbour Bombali virus, to other filoviruses: Ebola, Marburg, Taï Forest and Reston viruses. We demonstrate that only the bats inoculated with EBOV show high and disseminated viral replication and infectious virus shedding, without clinical disease, while the other filoviruses fail to establish productive infections. Notably, we evidence placental-specific tissue tropism and a unique ability of EBOV to traverse the placenta, infect and persist in foetal tissues of AFBs, which results in distinct genetic signatures of adaptive evolution. These findings not only demonstrate plausible routes of horizontal and vertical transmission in these bats, which are expectant of reservoir hosts, but may also reveal an ancillary transmission mechanism, potentially required for the maintenance of EBOV in small reservoir populations.
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Quirópteros , Ebolavirus , Fiebre Hemorrágica Ebola , Virus , Embarazo , Animales , Femenino , Placenta , Zoonosis , Replicación ViralRESUMEN
Giardia lamblia is a common parasitic protist that infects the small intestine and causes giardiasis, resulting in diarrhea, vomiting, weight loss, and malabsorption. Giardiasis leads to cellular damage, including loss of microvilli, disruption of tight junctions, impaired barrier function, enzyme inhibition, malabsorption, and apoptosis. In the host, motile Giardia trophozoites attach to the duodenal microvilli using a unique microtubule organelle called the ventral disc. Despite early observations of disc-shaped depressions in microvilli after parasite detachment, little is known about disc-mediated attachment mechanisms and there little direct evidence showing that parasite attachment causes cellular damage. However, advancements in in vitro organoid models of infection and genetic tools have opened new possibilities for studying molecular mechanisms of attachment and the impact of attachment on the host. Through high-resolution live imaging and a novel disc mutant, we provide direct evidence for disc contraction during attachment, resolving the long-standing controversy of its existence. Specifically, we identify three types of disc movements that characterize contraction, which in combination result in a decrease in disc diameter and volume. Additionally, we investigate the consequences of attachment and disc contractility using an attachment mutant that has abnormal disc architecture. In a human organoid model, we demonstrate that this mutant has a limited ability to break down the epithelial barrier as compared to wild type. Based on this direct evidence, we propose a model of attachment that incorporates disc contraction to generates the forces required for the observed "grasping" of trophozoites on the host epithelium. Overall, this work highlights the importance of disc contractility in establishing and maintaining parasite attachment, leading to intestinal barrier breakdown.
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AIMS: The objective of this study was to determine the effects of Ca-dipicolinic acid (CaDPA), cortex-lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. METHODS AND RESULTS: Bacillus subtilis spores, wild-type, CaDPA-less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine-treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild-type and CaDPA-less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l-1 dodecylamine in 4 h at 45°C with spores at ~108 ml-1 . Dodecylamine killing of decoated wild type and CLE-less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA-less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA-replete spores or dodecylamine-killed CLE-less spores peripherally, and cores of decoated CaDPA-less spores and dodecylamine-killed intact spores with CLEs. The IM of some decoated CaDPA-less spores was greatly reorganized. CONCLUSIONS: Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. SIGNIFICANCE AND IMPACT OF THE STUDY: This work gives new information on killing bacterial spores by dodecylamine, and how spore IM's relative impermeability is maintained.
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Aminas/farmacología , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Esporas Bacterianas/efectos de los fármacos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Mutación , Ácidos Picolínicos/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
High-pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high-pressure freezer. We explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for high-pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE-filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters were used for high-pressure freezing, freeze-substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms. LAY DESCRIPTION: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology-changes due to drying artefacts or the impact of chemical stabilisers, high-pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to -196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology-destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 µm in optimal cases). High-pressure freezing is regarded as the gold-standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high-pressure freezer and their transfer into the high-pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE-filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface-adhering higher cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters could be directly used for high-pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning-electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms.
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Bacterias/citología , Substitución por Congelación/métodos , Poliésteres/química , Biopelículas , Células HeLa , Humanos , PresiónRESUMEN
The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.
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Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Espectrometría Raman , Virión/ultraestructura , Silicio , Coloración y Etiquetado , Virus del Mosaico del Tabaco/ultraestructuraRESUMEN
AIMS: Successive application of negative staining transmission electron microscopy (TEM) and tip-enhanced Raman spectroscopy (TERS) is a new correlative approach that could be used to rapidly and specifically detect and identify single pathogens including bioterrorism-relevant viruses in complex samples. Our objective is to evaluate the TERS-compatibility of commonly used electron microscopy (EM) grids (sample supports), chemicals and negative staining techniques and, if required, to devise appropriate alternatives. METHODS AND RESULTS: While phosphortungstic acid (PTA) is suitable as a heavy metal stain, uranyl acetate, paraformaldehyde in HEPES buffer and alcian blue are unsuitable due to their relatively high Raman scattering. Moreover, the low thermal stability of the carbon-coated pioloform film on copper grids (pioloform grids) negates their utilization. The silicon in the cantilever of the silver-coated atomic force microscope tip used to record TERS spectra suggested that Si-based grids might be employed as alternatives. From all evaluated Si-based TEM grids, the silicon nitride (SiN) grid was found to be best suited, with almost no background Raman signals in the relevant spectral range, a low surface roughness and good particle adhesion properties that could be further improved by glow discharge. CONCLUSIONS: Charged SiN grids have excellent particle adhesion properties. The use of these grids in combination with PTA for contrast in the TEM is suitable for subsequent analysis by TERS. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports fundamental modifications and optimizations of the negative staining EM method that allows a combination with near-field Raman spectroscopy to acquire a spectroscopic signature from nanoscale biological structures. This should facilitate a more precise diagnosis of single viral particles and other micro-organisms previously localized and visualized in the TEM.
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Técnicas Microbiológicas/instrumentación , Microscopía Electrónica de Transmisión , Coloración Negativa , Compuestos de Silicona , Espectrometría Raman , Orthopoxvirus/aislamiento & purificaciónRESUMEN
The induction of angiogenesis is essential for successful engraftment of freely transplanted cells or cellular composites. How to augment angiogenesis to ensure an appropriate viability of the grafts is still under investigation. This study evaluated the proangiogenic capability of different syngeneic free liver transplants and elucidated the origin of the newly formed vascular network via use of an eGFP(+) /eGFP(-) (enhanced green fluorescent protein) cross-over design. Using intravital fluorescence microscopy, we found that neonatal and resected murine liver transplants implanted into dorsal skinfold chambers display a significantly enhanced vascularization compared to regular adult transplants. Immunohistochemically, less tissue hypoxia, apoptosis and macrophage infiltration was observed in the neonatal and resected transplants, which is in line with improved vascularization of those grafts. Additionally, electron microscopy revealed morphological hallmarks of liver cells. eGFP(+) liver transplants implanted on eGFP(-) recipients displayed vascular sprouting from the grafts themselves and connection to the recipients` microvasculature, which also undergoes transient proangiogenic response. This process is described as external inosculation, with microvessels exhibiting a chimeric nature of the endothelial lining. These data collectively show that proliferative stimulation is taking effect on angiogenic properties of free transplants and might provide a novel tool for modulating the revascularization of free grafts.
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Trasplante de Hígado/métodos , Hígado/irrigación sanguínea , Neovascularización Fisiológica/fisiología , Animales , Apoptosis , Proliferación Celular , Supervivencia de Injerto , Proteínas Fluorescentes Verdes/metabolismo , Hipoxia , Inmunohistoquímica , Inflamación/patología , Hígado/patología , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación/fisiología , Microscopía Electrónica , Microscopía Fluorescente , Neovascularización Patológica , Factores de TiempoRESUMEN
BACKGROUND: Early occurrence of small-fibre neuropathy (SFN) is a common feature of Fabry disease (FD) - an X-linked storage disorder caused by reduced activity of the α-galactosidase A (α-GAL). Although SFN may result from different disorders, the cause is often unclear. Therefore, we investigated the frequency of FD in patients with SFN of unknown aetiology. METHODS: Patients with idiopathic SFN, established by sensory quantitative testing and/or skin biopsy, were examined for mutations in the α-GAL gene. Where mutations in the α-GAL gene were identified, levels of globotriaosylceramide (Gb(3)) were measured in urine and blood and the α-GAL activity was evaluated. When new mutations were detected, a diagnostic work-up was performed as well as a Gb(3) accumulation in the skin, lyso-Gb(3) in blood and Gb(3)_24 in urine were proved. RESULTS: Twenty-four of 29 eligible patients were enrolled in the study. Mutations in the α-GAL gene were observed in five patients. A typical mutation for FD (c.424T>C, [C142R]) was detected in one patient. In four patients, a complex intronic haplotype within the α-GAL gene (IVS0-10C>T [rs2071225], IVS4-16A>G [rs2071397], IVS6-22C>T [rs2071228]) was identified. The relevance of this haplotype in the pathogenesis of FD remains unclear until now. However, these patients showed increased concentrations of Gb(3) and/or lyso-Gb(3), while no further manifestations for FD could be proved. CONCLUSIONS: Fabry disease should be considered in patients with SFN of unknown aetiology, and screening for FD should be included in the diagnostic guidelines for SFN. The significance of the intronic haplotype regarding SFN needs further evaluation.
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Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/epidemiología , Polineuropatías/genética , Adulto , Anciano , Análisis Mutacional de ADN , Enfermedad de Fabry/genética , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Mutación , Proyectos Piloto , alfa-Galactosidasa/análisis , alfa-Galactosidasa/genéticaRESUMEN
AIMS: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. METHODS AND RESULTS: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10-µl sample volume onto the sample support was 10(6) particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 µl, the detection limit could be reduced to 10(5) particles per ml for spores and to 5 × 10(4) particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. CONCLUSIONS: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. SIGNIFICANCE AND IMPACT OF THE STUDY: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.
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Bioterrorismo , Microscopía Electrónica/métodos , Bacillus subtilis/ultraestructura , Límite de Detección , Coloración Negativa , Poxviridae/ultraestructura , Esporas Bacterianas/ultraestructura , Virión/ultraestructuraRESUMEN
Lesion studies in animals are widely used forevaluating physiological parameters. However,changes in anatomical structures of the neuropilhave often been neglected in the interpretationof the physiological results. Our study showsthat anatomical data should also be considered.Here, the Fink-Heimer-method was used todemonstrate degeneration patterns after lesionsin the rat brain. In detail, the ascending noradrenergicfibres were incised at the level of thedorsal tegmental area of the mesencephalon.The resulting degeneration pattern correspondslargely with the noradrenergic projection. Theextent of degeneration is less than the physiologicalresults in the literature predict. The interruptionof the medial forebrain bundle at theposterolateral hypothalamic level led to a degenerationof the supraoptic decussation, where,according to physiological data, locus coeruleusfibres had been assumed to cross to the contralateralhemisphere. Beside axotomy, the lesionscaused edema, hemorrhage and necrosis.They were not restricted to the width of theblade and did not only affect the catecholaminergicsystem but led to degeneration inmediobasal forebrain sites where gonadotropinreleasinghormone-neurons are present, in fibresystems which carry afferents and efferents ofthese neurons and in hypothalamic and extrahypothalamicstructures that influence the release of the gonadotropin-releasing hormone. Thus,both anatomical and physiological aspects areimportant for a correct evaluation of lesion studiesin brain/behavior research (AU)
No disponible
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Ratas , Animales , Prosencéfalo/fisiopatología , Vías Nerviosas/lesiones , Degeneración Nerviosa/etiología , Radiocirugia/métodos , Neurópilo/fisiología , Fibras Adrenérgicas/fisiología , Modelos Animales de Enfermedad , Ratas Sprague-DawleyRESUMEN
A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.
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We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K(d), 10 nm) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates.
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Proteínas Portadoras/genética , Síndrome de Chediak-Higashi/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas/genética , Animales , Sitios de Unión/genética , Brefeldino A/farmacología , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Células COS , Proteínas Portadoras/metabolismo , Pollos , Clonación Molecular , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Citosol/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Proteínas del Helminto/genética , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Estructura Terciaria de Proteína/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Ratas , Homología de Secuencia de Aminoácido , Proteínas de Transporte VesicularRESUMEN
Antennae of Bombyx mori and Helicoverpa armigera larvae were immunolabelled with antisera raised against the pheromone-binding protein or the general odorant-binding protein 2 of Antheraea polyphemus to assign the expression of these proteins to individual sensilla and to compare the localization pattern with that in sensilla of adult moths. Specific labelling of antennal sensilla was only obtained with the antiserum against general odorant-binding protein 2. Among the few sensilla present on the antenna the three large sensilla basiconica, which are suspected to be olfactory in function, were labelled. These sensilla are compound sensilla consisting of several sensillum units which form a common sensory hair. The hair is single-walled and pierced by many pores. Labelling of sensillum compartments was the same as in sensilla of adults. Prominent labelling of the sensillum lymph is accompanied by labelling of secretory organelles in the two outermost auxiliary cells and of endocytotic pathways in all sensillum cells. The results suggest that general odorant-binding protein is expressed in single-walled multiporous sensilla of presumed olfactory function on the antenna of moth larvae. The overall identity of the localization pattern for general odorant-binding protein between larval and adult sensilla implies a similar role of these proteins in olfactory stimulus transduction.
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Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys(4) zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.
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Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Contráctiles , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/química , Terminales Presinápticos/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas Portadoras/genética , Línea Celular , Membrana Celular/metabolismo , Pollos , Proteínas del Citoesqueleto/genética , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos , Especificidad de Órganos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Profilinas , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Homología de Secuencia de Aminoácido , Dedos de Zinc , Proteína de Unión al GTP rab3A/metabolismoRESUMEN
Lipoproteins are plant-derived surface-active biopolymers, which act as emulsifying as well as viscosity-enhancing agents in oil-in-water emulsions. Depending on the degree of hydrolization, lipoproteins are dispersible or even soluble in water. In the presence of low to medium polar oils, lipoproteins are adsorbed and align at the oil-water interface, whereas in mixtures with high polar oils the lipoproteins are repelled from the oil-water interface. The water-dispersible lipoproteins show higher interfacial activity than the hydrolysates. Lipoproteins bear a negative electric charge in aqueous dispersions at pH 6.5, which is probably the reason for the stabilization of oil droplets against coalescence. Lipoprotein creams were characterized in terms of particle size, rheology, and emulsion stability against sedimentation, which was evaluated by a near-infrared sedimentometer. After topical application, emulsion stability breaks down and an emulsion film is formed on the skin surface. Lipoprotein creams cause a distinct increase in skin pliability and skin moisture and show excellent skin compatibility. In a home use test the panelists appreciated the cosmetic and caring properties of the lipoprotein cream.
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During aging, the epidermis and dermis become thin and an efficient anti-aging product should be able to stimulate the metabolism of senescent fibroblast and keratinocytes, in order to increase the quantity of extra-cellular matrix components such as collagen and glycosaminoglycans. A study performed in parallel on an in vitro skin equivalent model, and in vivo, with human volunteers, demonstrated the efficacy of one specific soya biopeptide for anti-aging properties. Such a biopeptide induces a significant increase of glycosaminoglycans synthesis in vitro and in vivo after a one-month treatment. We also showed that this new cosmetic ingredient is able to stimulate favourably the collagen synthesis in vitro and in vivo. This study provided the proof for anti-aging properties of a new soya biopeptide and also validated the skin equivalent model developed for this experimentation as an alternative method to animal or human testing for some cosmetic efficacy evaluations.
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Different odorant-binding proteins (OBPs) were isolated from total antennal homogenates of male and female Bombyx mori. Proteins were separated according to their isoelectric point by using preparative fast-flow isoelectrofocusing. Odorant-binding proteins were identified in immunoblots by antisera raised against the pheromone-binding protein (anti-PBP) and the general odorant-binding protein (anti-GOBP2) of Antheraea polyphemus. Four proteins cross-reacting with anti-PBP were detected in males and two in females, while three proteins cross-reacting with anti-GOBP2 were found in males and five in females. Both anti-PBP and anti-GOBP2 cross-reacting proteins had an apparent molecular weight of 15-16 kDa. In parallel, the same two antisera were used in immunocytochemical studies in order to determine the distribution of these proteins within the various subtypes of olfactory sensilla. The presence of multiple odorant-binding proteins within one moth species as well as their complex distribution pattern support the suggestion that soluble OBPs might have a function in odorant discrimination.
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Bombyx/metabolismo , Vías Olfatorias/metabolismo , Receptores Odorantes/metabolismo , Animales , Western Blotting , Bombyx/anatomía & histología , Reacciones Cruzadas , Femenino , Sueros Inmunes , Proteínas de Insectos/inmunología , Focalización Isoeléctrica , Masculino , Receptores Odorantes/inmunologíaRESUMEN
Electrophysiological in situ recordings from pheromone-sensitive sensilla trichodea of Bombyx mori males with a recording pipette which contained G-protein-activating fluoride, showed receptor cell activity similar to that evoked by pheromone stimulation. This suggests that G-proteins might be physiologically active in olfactory sensilla of insects in situ. Biochemical experiments using specific antibodies revealed the presence of G-protein, belonging to the Gq family, in antennal preparations. Similar G-protein was identified in sensory hair preparations of Antheraea pernyi which contained only cuticle, sensillum lymph and dendritic material. Moreover, the absence of this G-protein in pure sensillum lymph preparations indicates its association with the receptive dendrites. This particular association could be shown by immunolabelling studies at the ultrastructural level. Strong specific labelling of membranes of receptor-cell dendrites was found in all types of olfactory sensilla present on the antenna of the silkmoths. Additional specific labelling of apical membranes of auxiliary cells, epidermal cells and membranes forming the axon/glia interface demonstrated that this G-protein is not restricted to the sensory dendrites and that other signal-transduction pathways could be present at these membranes. In summary, the experiments imply a participation of G-protein of the Gq family in signal transduction of olfactory receptor cells in moths.