RESUMO
A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.
Assuntos
Surtos de Doenças/veterinária , Doenças dos Peixes/virologia , Infecções por Rhabdoviridae/epidemiologia , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/isolamento & purificação , Animais , Aquicultura , Efeito Citopatogênico Viral , Inglaterra , Ensaio de Imunoadsorção Enzimática , Peixes , Técnicas Histológicas , Imunoensaio , Rhabdoviridae/patogenicidade , Infecções por Rhabdoviridae/transmissão , Homologia de SequênciaRESUMO
Highly chemoselective amide forming ligation reactions have facilitated the synthetic access to proteins and other amide-linked bioconjugates. In order to generalize this approach, a N(alpha)-2-phenyl ethanethiol scaffold has been developed to promote S to N acyl transfer in a manner analogous to native chemical ligation with N-terminal cysteine residues. Analysis of scaffold-mediated ligation reactions in aqueous solution indicate that the ligation rate at Xaa-Gly junctions is sufficient for the synthesis of large polypeptides. In addition, it was found that the ligation rate is independent of the stereocenter in the scaffold and S- to N-acyl transfer is rate limiting. These studies indicate that the N(alpha)-2-phenyl ethanethiol scaffold is a good candidate for the development of a ligation chemistry for the formation of Xaa-Gly peptides and other unhindered amides.
Assuntos
Amidas/síntese química , Glicina/química , Métodos , Peptídeos/síntese química , Proteínas/síntese química , Compostos de Sulfidrila/químicaAssuntos
Apoproteínas/análise , Capsídeo/análise , Mioglobina/análise , Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Ribonuclease Pancreático/análise , Apoproteínas/metabolismo , Capsídeo/metabolismo , Mioglobina/metabolismo , Proteínas/análise , Ribonuclease Pancreático/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Termolisina/metabolismoRESUMO
When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.
Assuntos
Apresentação de Antígeno , Endopeptidases/isolamento & purificação , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microscopia de Fluorescência/métodos , Peptídeos/metabolismo , Transporte Biológico , Transferência de Energia , Corantes Fluorescentes/química , Peptídeos/síntese química , Rodaminas/química , Succinimidas/químicaRESUMO
A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N(alpha)-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N(alpha)-ethanethiol-containing peptide segment with a peptide-alphaCOSR segment. In this study, Nalpha-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nalpha-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K(a)) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The K(a) value for ((Nalpha-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.
Assuntos
Aprotinina/química , Dissulfetos/química , Sequência de Aminoácidos , Animais , Aprotinina/análogos & derivados , Bovinos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Inibidores de Serina Proteinase/síntese química , Tripsina/químicaRESUMO
The information that targets a protein to the nucleus often consists of a short cluster of basic amino acids called a nuclear localization sequence (NLS). Since a wide range of sequences rich in basic amino acid residues function as NLSs, we postulated that an NLS-like sequence composed exclusively of D-amino acids might have biological activity. We synthesized peptides corresponding to the c-Myc NLS composed of either all L or D-amino acids, both in the forward and reverse order. We tested these peptides for nuclear import activity in a digitonin-permeabilized cell assay. All four peptide-bovine serum albumin conjugates localized to the nucleus with similar efficiency, and each conjugate competed for import with an SV40 large T antigen-derived NLS conjugate. Cross-linking experiments with free NLS peptides in HeLa cytosol indicated that each peptide bound to a protein that migrated at the molecular weight of importin alpha. Recombinant importin alpha, importin beta, Ran, and NTF2 alone were sufficient to support the import of both L-form and D-form conjugates in permeabilized cells. This indicates that both D- and L-form NLS peptides use the same import machinery. Although the free D-forms of the NLS were proteolytically resistant in cytosol, the L-forms were rapidly degraded. To our knowledge, this is the first example of an intracellular pathway in which the receptor is insensitive to the chirality of the ligand.