RESUMEN
Nucleic acids catalyse the conversion of alpha-helical prion protein to the beta-structured protein oligomers and amyloids structurally similar to the infectious isoform of protein. Simultaneously, the prion protein, similar to gene regulating proteins, bends, unwinds and condenses nucleic acid. These properties might be related to the biological function and prion infection.
Asunto(s)
Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Priones/química , Priones/metabolismo , Animales , Humanos , Conformación Molecular , Enfermedades por Prión/terapia , Priones/genética , Unión ProteicaRESUMEN
Nucleic-acid-induced polymerisation of prion protein, when monitored by anilino naphthalene sulfonic acid dye, shows, successively, an immediate fluorescence increase of the dye upon mixing of the reactants, followed by a lag period in which the dye fluorescence remains unchanged, and then a phase in which dye fluorescence increases with time. The biological polyamines spermine and spermidine reduce the extent of the initial fluorescence increase, increase the lag period, and reduce both the rate and the extent of increase in fluorescence intensity of the dye in the final phase of the reaction. Spermidine is less effective than spermine in all of these processes. A nearly fivefold lower concentration of spermine can inhibit polymerisation of prion protein by tRNAs compared to the same process induced by double-stranded nucleic acid. The change in the secondary structure of the globular domain of the protein induced by nucleic acid is reversed by the addition of spermine, and it prevents structural destabilization of this domain induced by nucleic acids. It is suggested that physiological event(s) that would reduce the concentrations of intracellular biological amines may make nucleic acid available to induce oligomerization and polymerisation of cellular prion protein related to prion disease.
Asunto(s)
Poliaminas Biogénicas/farmacología , ADN/metabolismo , Priones/metabolismo , ARN de Transferencia/metabolismo , Espermidina/farmacología , Espermina/farmacología , Naftalenosulfonatos de Anilina/metabolismo , Biopolímeros , Dicroismo Circular , ADN/antagonistas & inhibidores , Colorantes Fluorescentes/metabolismo , Priones/química , Pliegue de Proteína , Estructura Secundaria de Proteína , ARN de Transferencia/antagonistas & inhibidores , TemperaturaRESUMEN
Nucleic acid induces conformational changes in the prion protein (23-231 amino acids) to a structure resembling its pathological isoform. The prion protein, in turn, facilitates DNA strand transfer and acts as a DNA chaperone which is modulated by the N-terminal unstructured basic segment of the protein. Here we have studied the prion protein induced conformational changes in DNA using oligonucleotides covalently labeled with the energy donor fluorescein and the acceptor rhodamine moieties by fluorescence resonance energy transfer (FRET) and by thermal stability of the unlabeled oligonucleotides. The protein induces a strong FRET effect in the oligonucleotides evidenced from the simultaneous quenching of fluorescence intensity of the donor and increase in the fluorescence intensity of the acceptor, which indicate bending of the oligonucleotides by the prion protein. The energy transfer efficiency induced by the protein is greater for the larger oligonucleotide. The prion protein also induces significant structural destabilization of the oligonucleotides observed from the lowering of their melting temperatures in the presence of the protein. The truncated globular prion protein 121-231 fragment neither induces FRET effect on the oligonucleotides nor destabilizes their structures, indicating that the N-terminal segment of the prion protein is essential for the DNA bending process. Equilibrium binding and kinetics of FRET show that the protein binding to the oligonucleotides and their bending occur simultaneously. The DNA structural changes observed in the presence of the prion protein are similar to those caused by proteins involved in initiation and regulation for protein synthesis.
Asunto(s)
Oligonucleótidos/química , Priones/química , Proteínas de Unión al ADN , Transferencia Resonante de Energía de Fluorescencia , Calor , Humanos , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Oligonucleótidos/metabolismo , Fragmentos de Péptidos/metabolismo , Priones/metabolismo , Priones/farmacologíaRESUMEN
The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.
Asunto(s)
Calor , Metilaminas/farmacología , Priones/química , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas Recombinantes/química , Naftalenosulfonatos de Anilina/metabolismo , Animales , Benzotiazoles , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Ratones , Muramidasa/química , Fragmentos de Péptidos/química , Priones/ultraestructura , Unión Proteica , Dispersión de Radiación , Solubilidad/efectos de los fármacos , Tiazoles/metabolismo , Triptófano/metabolismoRESUMEN
The infectious agent of transmissible spongiform encephalopathies (TSE) has been considered to be PrP(SC), a structural isoform of cellular prion protein PrP(C). PrP(SC) can exist as oligomers and/or as amyloid polymers. Nucleic acids induce structural conversion of recombinant prion protein PrP and PrP(C) to PrP(SC) form in solution and in vitro. Here, we report that nucleic acids, by interacting with PrP in solution, produce amyloid fibril and fibres of different morphologies, similar to those identified in the diseased brains. In addition, the same interaction produces polymer lattices and spherical amyloids of different dimensions (15-150 nm in diameters). The polymer lattices show apparent morphological similarity to the two-dimensional amyloid crystals obtained from linear amyloids isolated in vivo. The spherical amyloids structurally resemble "spherical particles" observed in natural spongiform encephalopathy (SE) and in scrapie-infected brains (TSE). We suggest that spherical amyloids, PrP(SC)-amylospheroids, are probable constituents of the coat of the spherical particles found in vivo and the latter can act as protective coats of the SE and TSE agents in vivo.
Asunto(s)
Amiloide/metabolismo , Ácidos Nucleicos/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Amiloide/ultraestructura , Animales , Ratones , Microscopía Electrónica , Priones/ultraestructura , Unión ProteicaRESUMEN
Structural change in the cellular prion protein, PrPC to a ProteinaseK-resistant beta-sheet-rich insoluble form PrPSC and its accumulation have been considered to be central to the pathogenesis of the prion diseases (TSE). In a recent paper, Deleault et al have shown that specific endogenous RNA molecules can induce in vitro structural conversion of endogenous PrPC to PrPSC. Small highly structured synthetic RNAs can also induce this conversion process. However, recent in vivo results show that PrPSC is not directly involved in the prion pathogenesis. It is possible, however, that nucleic-acid-induced PrPSC associated with the inducer nucleic acid could be the components of the infectious agent.
Asunto(s)
Enfermedades por Prión/etiología , Priones/metabolismo , Priones/patogenicidad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Ácidos Nucleicos/metabolismo , Priones/química , Conformación ProteicaRESUMEN
The unfolding of cellular prion protein and its refolding to the scrapie isoform are related to prion diseases. Studies in the literature have shown that structures of proteins, either acidic or basic, are stabilized against denaturation by certain neutral salts, for example, sulfate and fluoride. Contrary to these observations, the full-length recombinant prion protein (amino acid residues 23-231) is denatured by these protein structure stabilizing salts. Under identical concentrations of salts, the structure of the sheep prion protein, which contains a greater number of glycine groups in the N-terminal unstructured segment than the mouse protein, becomes more destabilized. In contrast to the full-length protein, the C-terminal 121-231 prion protein fragment, consisting of all the structural elements of the protein, viz., three alpha-helices and two short beta-strands, is stabilized against denaturation by these salts. We suggest that an increase in the concentration of the anions on the surface of the prion protein molecule due to their preferential interaction with the glycine residues in the N-terminal segment destabilizes the structure of the prion protein by perturbing the prion helix 1 which is the most soluble of all the protein alpha-helices reported so far in the literature. The present results could be relevant to explain the observed structural conversion of the prion protein by anionic nucleic acids and sulfated glycosaminoglycans.
Asunto(s)
Priones/química , Pliegue de Proteína , Sales (Química)/química , Secuencia de Aminoácidos , Animales , Tampones (Química) , Dicroismo Circular , Calor , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Muramidasa/química , Fragmentos de Péptidos/química , Desnaturalización Proteica , Estructura Secundaria de Proteína , Ovinos , Soluciones , Espectrometría de Fluorescencia , Sulfatos/química , Triptófano/químicaRESUMEN
The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.
Asunto(s)
Amiloide/química , Amiloide/metabolismo , ADN/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Priones/química , Priones/metabolismo , Pliegue de Proteína , Amiloide/ultraestructura , Naftalenosulfonatos de Anilina/metabolismo , Animales , Benzotiazoles , Biopolímeros/química , Biopolímeros/metabolismo , Birrefringencia , Dicroismo Circular , Rojo Congo/metabolismo , Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Microscopía Electrónica , Fragmentos de Péptidos/ultraestructura , Priones/ultraestructura , Unión Proteica , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Espectrometría de Fluorescencia , Temperatura , Tiazoles/metabolismo , Triptófano/metabolismoRESUMEN
Interaction between nucleic acid and recombinant murine prion protein, MoPrPC resulted in a time-dependent change in the nucleic acid morphology revealed by electron microscopy. After the addition of the protein to DNA, association of small number of nucleic acid molecules (nucleo-protein complex) was followed by aggregation of large number of them still retaining their initial linear morphology. With increase in the incubation time, ordered aggregation resulted in small condensed spherical globules. Subsequently, the formation of large condensed particles took place either by fusion of the already formed small globules or by accumulation of more nucleic acid molecules on them. The condensed nucleic acid structures observed here were different from other known morphologically altered nucleic acid structures induced by different cellular proteins. The condensed nucleic acid structures dissociated spontaneously. The formation of the prion protein-induced condensed nucleic acid structures resembled the human immunodeficiency virus 1 nucleocapsid protein NCp7-induced condensed ordered aggregates of nucleic acids. In the latter system, both the processes of condensation and dissociation of the nucleoprotein complex are believed to be responsible for the functional properties of the HIV-1 virus. Demonstration of functional activity of the prion protein-nucleic acid complex would be relevant for a role of nucleic acid in prion diseases.
Asunto(s)
ADN/metabolismo , ADN/ultraestructura , Proteínas PrPC/metabolismo , Proteínas PrPC/ultraestructura , Animales , Composición de Base , Citosina , ADN/química , Guanina , Cinética , Ratones , Microscopía Electrónica , Proteínas PrPC/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Recombinant prion protein has been used earlier to understand the structural properties of cellular prion protein PrP(C) and to understand conformational change of PrP(C) to its isoform, PrP(Sc) which is believed to be responsible for the prion disease. Here we report that murine recombinant prion protein, MoPrP(C) polymerizes in the presence of nucleic acid. The aggregation process and the properties of the aggregates have been monitored by physical, biochemical and ultrastructural studies. An increase in the turbidity at 0,90 degrees light scattering is observed when the protein is added to nucleic acid. An increase in the fluorescence of anilino naphthalene sulfonic acid dye (ANS) accompanying a blue shift in its emission maxima is observed when the aggregate obtained from prion protein and DNA reaction is added to it. The kinetics of the increase of the ANS fluorescence during aggregation process show lag periods which depend linearly on the nucleic acid concentration but show a biphasic dependence on the protein concentration. The change in the fluorescence properties of the dye in the presence of the aggregates obtained in the present study and in the presence of the protein PrP 27-30 amyloid isolated in vivo reported in literature are similar. The dye Congo Red binds to the aggregates resulting from the aggregation reaction.The ultrastructural analysis revealed polymeric structures with amyloid like morphologies and smaller oligomeric structures. In addition, condensed nucleic acid structures are also observed which are morphologically different from histone induced condensed nucleic acid structures but are similar to Human Immunodeficiency Virus-1 nucleocapsid protein, NCp7, induced nucleic acid structures. The aggregates show resistance to degradation by proteinase K treatment. Charge neutralization resulting from the MoPrP(C)-DNA interaction and accompanying structural changes in the molecules may explain the observed effects.
Asunto(s)
ADN/química , Proteínas PrPC/química , Naftalenosulfonatos de Anilina , Animales , Colorantes , Rojo Congo , Colorantes Fluorescentes , Cinética , Ratones , Microscopía Electrónica , Polímeros/química , Proteínas Recombinantes/química , Espectrometría de FluorescenciaRESUMEN
The human prion peptide PrP106-126 polymerizes in the presence of DNA both in its circular and linearized forms under solution conditions where the peptide alone does not polymerize. The polymerization process has been monitored by the increase in the fluorescence of anilino naphthalene sulfonic dye which detects the availability of the hydrophobic surface(s) in the aggregate as a consequence of polymerization. The polymerization is a nucleation dependent phenomenon as is evidenced from an existence of a lag period before the onset of the polymerization and a strong dependence of the polymerization on the prion peptide concentrations. The reaction is dependent on the pH as seen from rapid polymerization at pH 5 compared to the reaction at neutral pH where no polymerization is observed after a relatively long period of incubation. The polymer has been characterized as amyloid by using new absorbing and emitting species resulting from the interaction of the polymer with the amyloid specific fluorescent dye, Thioflavine S. This is probably the first demonstration that an endogenous macromolecule can influence the polymerization of a prion peptide. We have previously shown that there is a conformational change in the nucleic acid as a consequence of this interaction. This prion peptide is considered as a model to understand prion diseases as is evidenced from its toxicity towards primary brain cells in culture. The peptide encompasses one of the important amyloidogenic regions of the normal cellular prion protein. Demonstration of nucleic acid induced polymerization of the normal and scrapie prion isoforms accompanying a change in the nucleic acid conformation can establish a possible role of nucleic acid in prion disease.
Asunto(s)
Amiloide/química , ADN/química , Fragmentos de Péptidos/química , Priones/química , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina , Animales , Benzotiazoles , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Polímeros/química , Enfermedades por Prión/etiología , Unión Proteica , Soluciones , TiazolesRESUMEN
Recombinant porcine interferon gamma (IFN gamma) at neutral pH is characterized by a tryptophan (Trp) fluorescence maximum around 343 nm and a rigid conformation, evidenced from tryptophan polarization values. Guanidine HCl shifts the protein emission spectra further to the red and decreases the fluorescence polarization values, indicating denatured IFN gamma in these solutions. In acidic solutions (3 < pH < 4), the emission spectra show a blue shift and lower tryptophan polarization. The midpoint of transition of these fluorescence properties occurs around pH 3.5-3.6. The protein in NaClO4 solution at neutral pH is similarly characterized by a blue shift in the tryptophan fluorescence maxima and low polarization values. The extent of quenching of tryptophan fluorescence by acrylamide is less in acid and in NaClO4 solutions of IFN gamma compared to its native form. This indicates a lower accessibility of the tryptophan in the altered conformation of the protein. The emission spectra of IFN gamma in NaClO4 solution shows a decrease in the tryptophan fluorescence intensity with simultaneous shift of the emission spectra over time. The presence of two conformational forms of IFN gamma in perchlorate solution is evidenced from an isofluorescent point at 315 nm. The change in the conformational state in perchlorate solution is characterized by first order kinetics. The dye anilinonaphthalene sulfonic acid does not bind either to the native IFN gamma or to its denatured form. However, the dye binds to the acid form of IFN gamma, as well as when the protein is present in NaClO4 solution at neutral pH. These observations, together with the results from literature that IFN gamma retains its secondary structure in acid solution to a considerable degree, would suggest that the protein exists as a molten globule-like state in acidic solution. Similarities of the protein fluorescence and 1-anilino-8-naphthalene-sulfonic-acid (ANS) binding properties of the protein in NaClO4 and acid solutions indicate that IFN gamma also exists in a molten globule-like state in perchlorate solution at neutral pH.
Asunto(s)
Ácidos/química , Interferón gamma/química , Percloratos/química , Compuestos de Sodio/química , Naftalenosulfonatos de Anilina/química , Animales , Concentración de Iones de Hidrógeno , Interferón gamma/metabolismo , Unión Proteica , Soluciones , Espectrometría de Fluorescencia , Porcinos , TriptófanoRESUMEN
Synthetic prion peptide PrP106-126 has been used as a model to understand prion diseases. The conformation of the peptide depends on the environmental conditions and it forms amyloid in vitro. The potential of this prion peptide to interact with nucleic acids has been studied using a fluorescent labelled nucleic acid by kinetic and equilibrium methods. A decrease in the fluorescence of the labelled DNA induced by the peptide with time is observed which is pH, ionic strength and temperature dependent. The activation energy of the reactions is approximately 100 kJ mol-1. Lysine tripeptide and spermidine, carrying the same number of positive charges as the prion peptide, do not show an appreciable effect on the DNA. The binding constant between the prion peptide and DNA has a value of > 10(6) M-1 in phosphate buffer, pH 8 which is of the same order of magnitude as the binding of a retroviral protein, p10, with model nucleic acids. It is tempting to speculate that this interaction might play a role in the prion diseases.
Asunto(s)
Papillomavirus Bovino 1/genética , ADN Viral/metabolismo , Fragmentos de Péptidos/metabolismo , Priones/metabolismo , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Concentración OsmolarRESUMEN
In a number of neurodegenerative diseases, a change in the conformation of specific proteins occurs which is considered to be related to the development of the disease process. In general, a change in the protein conformation from a predominantly alpha-helical to a predominantly beta-sheet structure, due to genetic or environmental factors, leads to oligomerization of the proteins. In Alzheimer's and prion diseases, insoluble precipitates or plaques are often observed in vivo as a consequence of beta-fibril formation due to the polymerization of the proteins. In certain other neurodegenerative diseases, for example in Huntington disease, the beta-sheet oligomers may interact differently with other functional proteins compared to the monomeric proteins inducing the disease. Biophysical studies would provide information regarding the forces responsible for the oligomerization of these proteins and hence provide methods for detecting these diseases, leading eventually to therapeutic approaches.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades por Prión/metabolismo , Estructura Secundaria de Proteína , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Animales , Humanos , Sustancias Macromoleculares , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/veterinaria , Mutación Puntual , Enfermedades por Prión/genética , Enfermedades por Prión/veterinaria , Priones/química , Priones/genéticaRESUMEN
A xenotropic Moloney murine leukemia virus-derived recombinant retrovirus (MMuLVSVnlslacZ) has been utilized to study the mechanism of virus entry into endothelial and epithelial porcine cells. In the genome of this recombinant retrovirus, the nlslacZ reporter gene is under the transcriptional control of both LTR and SV40 early promoter. The entry of the retrovirus has been determined from the expression of this transduced reporter gene after its integration into the infected cells. This allows the detection of a very low level of viral infection and hence entry of the virus. Exposure of the virus-cell mixture to acidic pH (less than 6) during the early phase of interaction reduces the level of internalization. Cellular infection in presence of weak bases, ammonium chloride and amantadine and an ionophore monensin at concentrations sufficient to neutralize the endosomal pH does not modify the extent of viral entry into the cells. The results indicate that the entry of the recombinant retrovirus into porcine cells takes place by a pH-independent viral membrane-cell plasma membrane fusion mechanism.
Asunto(s)
Leucemia Experimental/metabolismo , Virus de la Leucemia Murina de Moloney/crecimiento & desarrollo , Amantadina/farmacología , Cloruro de Amonio/farmacología , Animales , Células Cultivadas , Concentración de Iones de Hidrógeno , Operón Lac/genética , Leucemia Experimental/patología , Virus de la Leucemia Murina de Moloney/patogenicidad , Monensina/farmacología , Proteínas Recombinantes/biosíntesis , Porcinos , Transducción Genética , Replicación Viral/fisiología , beta-Galactosidasa/biosíntesisRESUMEN
Swine testis (ST) cell lines producing a murine recombinant retrovirus (RRV) were established in order to transfer the bacterial lacZ gene fused to a nuclear location signal (nlslacZ) into animal cells. ST cells were infected with the supernatant of the cat G355.5LacZ2 cell line which produces amphotropic and xenotropic MMuLVSVnlslacZ-defective RRV and wild amphotropic and xenotropic MMuLV. Expression of the nlslacZ reporter gene was under the transcriptional control of both the SV40 early promoter and the retroviral LTR. ST cells expressing the reporter gene were sorted and cloned by limiting dilutions. Fourteen STLacZ-cell lines were isolated and subsequently tested for virus production. Depending on the host range of the retroviruses, two cell lines (STBF11 and STAA3) produced both a xenotropic recombinant pseudotype and wild retroviruses; another (STAB 10) produced both an amphotropic recombinant pseudotype and wild retroviruses. Southern blot analysis of the producer cell lines was carried out to verify proviral integration. The efficiency of the different pseudotypes in the transfer of the nlslacZ reporter gene to cultured animal cells, including porcine cells, was compared to the pseudotyped RRV produced by cat lines. Our results showed that the xenotropic RRV produced by the porcine STBF 11 cell line has a high titre for cells from different species and led to a higher number of porcine endothelial and lymphoblastoid cells expressing the reporter gene than did RRV produced by the cat packaging cell lines.
Asunto(s)
Operón Lac , Recombinación Genética , Retroviridae/genética , Transfección , Animales , Gatos , Bovinos , Línea Celular , Separación Celular , Clonación Molecular , Endotelio , Vectores Genéticos , Cabras , Caballos , Linfocitos , Masculino , Ratones , Virus de la Leucemia Murina de Moloney/genética , Conejos , Retroviridae/crecimiento & desarrollo , Infecciones por Retroviridae/genética , Ovinos , Porcinos , Testículo , Integración Viral/genética , Replicación ViralRESUMEN
Exposure of broad bean (Victa faba L.) plants to 270 +/- 32 and 670 +/- 45 micrograms m 3SO2 for 1.5 hr daily between 40 and 85 days of their ages resulted in an increase in their transpiration rate, water saturation deficit, phenol content, and peroxidase activity and a decrease in protein content. With the increase in number of exposures of plants to SO2, chlorotic and brown, necrotic visible injury signs were also developed in leaves. It was further noted that the magnitude of undesirable biochemical changes, which possibly helped in the formation of new pigment characteristic of necrotic tissue of SO2-exposed plants, was not totally dependent on the pollutant concentration.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Fabaceae/efectos de los fármacos , Plantas Medicinales , Dióxido de Azufre/toxicidad , Envejecimiento/metabolismo , Análisis de Varianza , Fabaceae/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Peroxidasas/metabolismo , Fenoles/metabolismo , Proteínas de Plantas/metabolismo , Agua/análisisRESUMEN
Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.
Asunto(s)
Fluoresceína-5-Isotiocianato/análogos & derivados , Liposomas/metabolismo , Membrana Nuclear/metabolismo , Adsorción , Animales , Dextranos , Electroquímica , Transferencia de Energía , Fluoresceínas , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Hígado/ultraestructura , Fusión de Membrana , Ratones , Microscopía Fluorescente , Ácidos Fosfatidicos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Rodaminas/metabolismo , Espectrometría de FluorescenciaRESUMEN
The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.
Asunto(s)
Encéfalo/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Endosomas/fisiología , Liposomas/metabolismo , Fusión de Membrana , Animales , Bovinos , Fraccionamiento Celular , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Electroquímica , Transferencia de Energía , Fluoresceínas , Colorantes Fluorescentes , Técnica de Fractura por Congelación , Cinética , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica , Ácidos Fosfatidicos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Rodaminas/metabolismo , Espectrometría de FluorescenciaRESUMEN
DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.