RESUMO
We implement a massively parallel population Monte Carlo approximate Bayesian computation (PMC-ABC) method for estimating diffusion coefficients, sizes and concentrations of diffusing nanoparticles in liquid suspension using confocal laser scanning microscopy and particle tracking. The method is based on the joint probability distribution of diffusion coefficients and the time spent by a particle inside a detection region where particles are tracked. We present freely available central processing unit (CPU) and graphics processing unit (GPU) versions of the analysis software, and we apply the method to characterize mono- and bidisperse samples of fluorescent polystyrene beads.
RESUMO
An overview is given on the development of technologies to allow reverse genetics of RNA viruses, i.e., the rescue of viruses from cDNA, with emphasis on nonsegmented negative-strand RNA viruses (Mononegavirales), as exemplified for measles virus (MV). Primarily, these technologies allowed site-directed mutagenesis, enabling important insights into a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted to (a) allow localization of virus replication in vivo through marker gene expression, (b) develop candidate multivalent vaccines against measles and other pathogens, and (c) create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses proposed as vectors, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favorably complemented.
Assuntos
Engenharia Genética , Vacina contra Sarampo/imunologia , Vírus do Sarampo/genética , Sarampo/imunologia , Animais , Genoma Viral , Humanos , Sarampo/prevenção & controle , Sarampo/virologia , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/genética , Vírus do Sarampo/imunologia , Mutagênese Sítio-Dirigida , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A new method, MUNIN (Multi-dimensional NMR spectra interpretation), is introduced for the automated interpretation of three-dimensional NMR spectra. It is based on a mathematical concept referred to as three-way decomposition. An NMR spectrum is decomposed into a sum of components, with each component corresponding to one or a group of peaks. Each component is defined as the direct product of three one-dimensional shapes. A consequence is reduction in dimensionality of the spectral data used in further analysis. The decomposition may be applied to frequency-domain or time-domain data, or to a mixture of these. Features of MUNIN include good resolution in crowded regions and the absence of assumptions about line shapes. Uniform sampling of time-domain data, a prerequisite for discrete Fourier transform, is not required. This opens an avenue for the processing of NMR data that do not follow oscillating behaviour, e.g. from relaxation measurements. The application of MUNIN is illustrated for a 1H-15N-NOESY-HSQC, where each component is defined as the set of all NOE peaks formed by a given amide group. As a result, the extraction of structural information simply consists of one-dimensional peak picking of the shape along the NOE-axis obtained for each amide group.
Assuntos
Algoritmos , Interpretação Estatística de Dados , Imageamento Tridimensional , Ressonância Magnética Nuclear Biomolecular/métodos , Artefatos , Análise de Fourier , Análise dos Mínimos QuadradosRESUMO
The backbone dynamics of the channel-forming peptide antibiotic zervamicin IIB (Zrv-IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. 'Model-free' analysis of the relaxation data showed that the peptide is fairly rigid on a sub-nanosecond time-scale. The residues from the polar side of Zrv-IIB helix are involved in micro-millisecond time-scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv-IIB ion channel formation.
Assuntos
Antibacterianos/química , Canais Iônicos/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos , Anisotropia , Simulação por Computador , Metanol/química , Conformação Molecular , Isótopos de Nitrogênio , Peptaibols , RotaçãoRESUMO
Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.
Assuntos
Antígenos CD/fisiologia , Macrófagos/fisiologia , Sarampo/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Interferon/fisiologia , Animais , Células Dendríticas/virologia , Humanos , Macrófagos/virologia , Vírus do Sarampo/fisiologia , Proteína Cofatora de Membrana , Camundongos , Monócitos/fisiologia , Monócitos/virologia , Receptor de Interferon alfa e beta , Replicação ViralRESUMO
We have genetically engineered a panel of recombinant measles viruses (rMVs) that express from various positions within the MV genome either the HN or F surface glycoproteins of mumps virus (MuV) or the env, gag or pol proteins from simian immunodeficiency virus (SIV). All rMVs were rescued from the respective antigenomic plasmid constructs; progeny viruses replicated comparably to the progenitor Edmonston B MV, but showed slight propagation retardation, which was dependent on the size and nature of the expressed proteins and on the genomic position of the inserts. All transgenes except that encoding mumps F glycoprotein were faithfully maintained and expressed even after virus amplification by 10(20). Our results suggest possible applications of rMVs as live-attenuated, multivalent vaccines against retroviruses such as SIV and HIV as well as other pathogens more distantly related to MV than MuV.
Assuntos
Antígenos Virais/genética , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Vírus da Caxumba/genética , Vírus da Caxumba/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Sequência de Bases , Chlorocebus aethiops , Primers do DNA/genética , Expressão Gênica , Genes Virais , Engenharia Genética , Vetores Genéticos , Vírus do Sarampo/crescimento & desenvolvimento , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Recombinação Genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
MUNIN (Multidimensional NMR Spectra Interpretation), a recently introduced approach exploiting the mathematical concept of three-way decomposition, is proposed for separation and quantitative relaxation measurements of strongly overlapped resonances in sets of heteronuclear two-dimensional spectra that result from typical relaxation experiments. The approach is general and may also be applied to sets of two-dimensional spectra with arbitrary modulation along the third dimension (e.g., J-coupling, diffusion). Here, the method is applied for the analysis of 15N rotating frame relaxation data.
Assuntos
Proteínas do Capsídeo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Azurina/química , Capsídeo/química , Interpretação Estatística de Dados , Proteínas de Membrana/química , Conformação Proteica , RotaçãoRESUMO
Subacute sclerosing panencephalitis (SSPE) is a progressive degenerative disease of the brain uniformly leading to death. Although caused by measles virus (MV), the virus recovered from patients with SSPE differs from wild-type MV; biologically SSPE virus is defective and its genome displays a variety of mutations among which biased replacements of many uridine by cytidine resides primarily in the matrix (M) gene. To address the question of whether the SSPE MVs with M mutations are passive in that they are not infectious, cannot spread within the CNS, and basically represent an end-stage result of a progressive infection or alternatively SSPE viruses are infectious, and their mutations enable them to persist and thereby cause a prolonged neurodegenerative disease, we utilized reverse genetics to generate an infectious virus in which the M gene of MV was replaced with the M gene of Biken strain SSPE MV and inoculated the recombinant virus into transgenic mice bearing the MV receptor. Our results indicate that despite biased hypermutations in the M gene, the virus is infectious in vivo and produces a protracted progressive infection with death occurring as long as 30 to 50 days after that caused by MV. In primary neuron cultures, the mutated M protein is not essential for MV replication, prevents colocalization of the viral N with membrane glycoproteins, and is associated with accumulation of nucleocapsids in cells' cytoplasm and nucleus.
Assuntos
Vírus SSPE/metabolismo , Panencefalite Esclerosante Subaguda/virologia , Proteínas da Matriz Viral/fisiologia , Animais , Células Cultivadas , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/citologia , Neurônios/virologia , Proteínas do Nucleocapsídeo/metabolismo , Vírus SSPE/genética , Vírus SSPE/crescimento & desenvolvimento , Vírus SSPE/fisiologia , Panencefalite Esclerosante Subaguda/mortalidade , Frações Subcelulares , Células Vero , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
In polarized epithelial cells measles virus (MV) is predominantly released at the apical cell surface, irrespective of the sorting of its two envelope glycoproteins F and H. It has been reported previously that the viral matrix (M) protein modulates the fusogenic capacity of the viral envelope glycoproteins. Here, extant MV mutants and chimeras were used to determine the role of M protein in the transport of viral glycoproteins and release of progeny virions in polarized epithelial CaCo2 cells. In the absence of M, envelope glycoproteins are sorted to the basolateral surface, suggesting that they possess intrinsic basolateral sorting signals. However, interactions of M with the glycoprotein cytoplasmic tails allow M-glycoprotein co-segregation to the apical surface, suggesting a vectorial function of M to retarget the glycoproteins for apical virion release. Whereas this may allow virus airway shedding, the intrinsic sorting of the glycoproteins to the basolateral surface may account for systemic host infection by allowing efficient cell-cell fusion.
Assuntos
Polaridade Celular , Células Epiteliais/virologia , Glicoproteínas/metabolismo , Vírus do Sarampo/fisiologia , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Animais , Células CACO-2 , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imunofluorescência , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Vírus do Sarampo/genética , Vírus do Sarampo/metabolismo , Fusão de Membrana , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/genética , Replicação ViralRESUMO
OBJECTIVE: To determine the effects of a low-dose mifepristone regimen on endometrium in the rhesus monkey by endometrial staging and analysis of molecular markers of endometrial receptivity. DESIGN: A prospective, randomized comparative study. SETTING: Academic research environment. ANIMAL(S): Normally cycling rhesus (Macaca mulatta) monkeys. INTERVENTION(S): Monkeys (5 per control or treatment group) received 0.03 mg of mifepristone in vehicle (sesame oil) per kilogram of body weight or vehicle daily from day 2 of the menstrual cycle to 7 days after the midcycle E2 surge. MAIN OUTCOME MEASURE(S): Serum estradiol (E2) and progesterone (P) levels; endometrial staging and immunoreactivity of leukemia inhibitory factor and interleukin-6 performed on fixed endometrial tissues; and relative abundance of endometrial estrogen and P receptor mRNA evaluated with semiquantitative reverse transcriptase polymerase chain reaction in which cyclophilin mRNA, a housekeeping gene product, was coamplified as the reference standard. RESULT(S): Mifepristone at 0.03 mg/kg/d induced a delay in the endometrial cycle with a shift from the late to midsecretory phase. This treatment regimen did not suppress the midcycle gonadotropin surge or, presumably, ovulation because P levels were normal during the midluteal phase. The staining intensity of leukemia inhibitory factor and interleukin-6 was dependent upon the endometrial stage and was decreased in treated monkeys. E and P receptor mRNAs increased significantly with mifepristone treatment compared with controls, another indication of delayed uterine staging. CONCLUSION(S): Mifepristone at 0.03 mg/kg/d had no antiovulatory effect but delayed development of the endometrium from the late to midsecretory phase. This study provides further evidence that endometrial maturation can be altered without affecting ovarian cyclicity.
Assuntos
Abortivos Esteroides/farmacologia , Endométrio/efeitos dos fármacos , Mifepristona/farmacologia , Abortivos Esteroides/administração & dosagem , Animais , Estradiol/sangue , Feminino , Imuno-Histoquímica , Macaca mulatta , Mifepristona/administração & dosagem , Peptidilprolil Isomerase/genética , Reação em Cadeia da Polimerase , Progesterona/sangue , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Padrões de ReferênciaRESUMO
Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.
Assuntos
Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Tripsina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Fusão Celular/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Primers do DNA/genética , Humanos , Sarampo/etiologia , Sarampo/patologia , Sarampo/virologia , Vírus do Sarampo/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Recombinação Genética , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/fisiologia , Virulência/efeitos dos fármacos , Virulência/genética , Virulência/fisiologiaRESUMO
The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.
Assuntos
Vírus Defeituosos/fisiologia , Sistema Linfático/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Sarampo/patologia , Animais , Antígenos CD/análise , Antígenos CD/genética , Encéfalo/patologia , Vírus Defeituosos/genética , Células Gigantes , Humanos , Imuno-Histoquímica , Hibridização In Situ , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Sistema Linfático/patologia , Macrófagos/virologia , Sarampo/virologia , Vírus do Sarampo/classificação , Vírus do Sarampo/isolamento & purificação , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Receptores de Interferon/análise , Receptores de Interferon/genética , Replicação ViralRESUMO
The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90-230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered "tail." The globular domain contains three alpha-helices comprising the residues 144-154, 173-194, and 200-228 and a short anti-parallel beta-sheet comprising the residues 128-131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219-228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219-226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.
Assuntos
Príons/química , Animais , Clonagem Molecular , Cricetinae , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
The measles virus (MV) P gene encodes three proteins: the P protein and two nonstructural proteins, C and V. Because the functions of both the C and V protein are unknown, we used MV C (C-) and V (V-) deletion recombinants generated by the MV reverse genetics system (F. Radecke, P. Spielhofer, H. Schnieder, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M. A. Billeter 1995. EMBO J. 14, 5773-5784). Compared to parental vaccine strain, Edmonston (Ed) MV, both had normal growth and cytopathic effects in Vero cells and showed similar growth kinetics in human neuroblastoma SK-N-MC cells and in primary mouse neurons expressing the MV receptor, CD46. However, in vivo, using YAC-CD46 transgenic mice as a model for MV induced CNS disease (M. B. A. Oldstone, H. Lewicki, D. Thomas, A. Tishon, S. Dales, J. Patterson, M. Manchester, D. Homann, D. Naniche, and A. Holz 1999. Cell 98, 629-640), C- and V- viruses differed markedly from wt Ed(V(+)C(+)) virus. Newborn mice inoculated with as little as 10(3) PFU of Ed strain became ill and died after 10-15 days. In contrast, those inoculated with 10(3) or 10(4) PFU of MV C- or MV V- showed significantly fewer and milder clinical symptoms and had a lower mortality. A total of 10(5) PFU V- virus were required to kill most YAC-CD46 mice, and less than half (44%) were killed with a corresponding dose of MV C-. Immunohistochemical staining for MV antigens showed similar extents of spread for MV C- and MV Ed but restricted spread for MV V- throughout the brain. Viral load and transcription were markedly reduced for V- but not for C-. Multiple cytokines and chemokines were equivalently upregulated for all three viruses. Therefore, MV C and V proteins encode virulence functions in vivo and likely operate via separate mechanisms.
Assuntos
Vírus do Sarampo/patogenicidade , Fosfoproteínas/fisiologia , Proteínas não Estruturais Virais/fisiologia , Proteínas Virais/fisiologia , Animais , Sequência de Bases , Encéfalo/virologia , Linhagem Celular , Chlorocebus aethiops , Primers do DNA/genética , Deleção de Genes , Genes Virais , Humanos , Sarampo/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios/virologia , Fosfoproteínas/genética , RNA Viral/genética , Recombinação Genética , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Virulência/genética , Replicação ViralRESUMO
In an open-label Swiss multicentre trial to assess patient acceptance, efficacy and safety of zolmitriptan 2.5 mg tablets in the acute treatment of migraine, 112 patients were enrolled and 281 migraine attacks evaluated. Patients completed a specially designed protocol during and after each attack. Special attention was given to the patients' subjective comparison of the study drug with previously used abortive treatments. On this matter, zolmitriptan performed clearly better than analgesics and NSAIDS (78% "better"). Compared with sumatriptan, zolmitriptan was narrowly preferred (45% "better", 36% "worse"). Headache intensity was reduced from moderate or severe to mild or none after one hour in 51% and after 2 hours in 70%. Median time to efficacy was 50 minutes. Autonomic symptoms such as nausea and photo- and phonophobia were improved in 45 to 55%. A subjectively significant improvement in general condition (functionality) was reported in 51% after one hour and in 72% after 2 hours. Headache recurrence was reported by 25% of the responders after a median time lapse of 14 hours. Adverse effects were reported by 22%, and were all short lasting and spontaneously reversible. Rapid efficacy, high and persistent response rate, improvement of headache, autonomic symptoms and general condition, and excellent tolerability are thus the reasons for the patients' preference of zolmitriptan over traditional abortive drugs.
Assuntos
Transtornos de Enxaqueca/tratamento farmacológico , Oxazolidinonas/uso terapêutico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxazolidinonas/efeitos adversos , Medição da Dor , Recidiva , Resultado do Tratamento , TriptaminasRESUMO
In humans, familial forms of transmissible spongiform encephalopathies (TSE; "prion diseases") have been shown to segregate with the exchange of individual amino acids in the prion protein (PrP) sequence. We used the NMR structure of the globular domain of mouse PrP in the cellular form (PrP(C)) as a starting point for investigations by long-time molecular dynamics (MD) simulations at ambient temperature of likely impacts of such mutations on the PrP(C) structure, making use of the fact that species-related amino acid replacements between mouse PrP and human PrP are spatially well separated from the disease-related mutations in human PrP. In the MD simulations these amino acid substitutions were found to have a variety of different effects on the protein structure, with some species showing altered packing of regular secondary structure elements, while other mutants showed no or only strictly localized changes of the structure near the variant amino acid. The fact that some of the disease-related amino acid exchanges cause no measurable change of the PrP(C) structure indicates that their influence on the conformational transition to the scrapie form of PrP may be due to modified intermolecular interactions during the aggregation process.
Assuntos
Substituição de Aminoácidos , Proteínas PrPC/química , Proteínas PrPC/genética , Doenças Priônicas/etiologia , Estrutura Terciária de Proteína , Animais , Simulação por Computador , Humanos , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , TemperaturaRESUMO
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Assuntos
Astrócitos/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Animais , Astrocitoma , Linhagem Celular , Imunofluorescência , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Proteínas Nucleares , Fatores de Transcrição , Proteína do Homeodomínio de Antennapedia , Isótopos de Carbono , Proteínas de Ligação a DNA/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Isótopos de Nitrogênio , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Estrutura Secundária de ProteínaRESUMO
Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.
Assuntos
Encéfalo/virologia , Hemaglutininas Virais/fisiologia , Vírus do Sarampo/patogenicidade , Adaptação Biológica , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Genes Virais , Vetores Genéticos , Células HeLa , Hemaglutininas Virais/genética , Humanos , Sarampo/patologia , Sarampo/virologia , Vacina contra Sarampo , Vírus do Sarampo/genética , Vírus do Sarampo/crescimento & desenvolvimento , Vírus do Sarampo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Roedores , Células Vero , VirulênciaRESUMO
A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of approximately 11 A and approximately 8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.