RESUMEN
Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk. The BRET-based biosensor is highly selective for bacterial protease activity compared with other proteases tested, notably including plasmin, which is abundant in milk. It incorporates a novel peptide linker that is selectively cleaved by P. fluorescens AprX proteases. The peptide linker is flanked by green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the linker by bacterial proteases from Pseudomonas fluorescens strain 65, leads to a 95% decrease in the BRET ratio. We applied an azocasein-based calibration method to the AprX biosensor using standard international enzyme activity units. In a 10-min assay, the detection limit for AprX protease activity in buffer was equivalent to 40 pg/mL (≈0.8 pM, 22 µU/mL) and 100 pg/mL (≈2pM, 54 µU/mL) in 50% (v/v) full fat milk. The EC50 values were 1.1 ± 0.3 ng/mL (87 µU/mL) and 6.8 ± 0.2 ng/mL (540 µU/mL), respectively. The biosensor was approximately 800x more sensitive than the established FITC-Casein method in a 2-h assay, the shortest feasible time for the latter method. The protease biosensor is sensitive and fast enough to be used in production settings. It is suitable for measuring bacterial protease activity in raw and processed milk, to inform efforts to mitigate the effects of heat-stable bacterial proteases and maximise the shelf-life of dairy products.
Asunto(s)
Péptido Hidrolasas , Pseudomonas fluorescens , Animales , Leche , Transferencia de Energía , Proteolisis , Proteínas Fluorescentes VerdesRESUMEN
Förster resonance energy transfer (RET) is the nonradiative transfer of energy from a donor to an acceptor fluorophore. The Förster distance (R(0)), being the fluorophore separation corresponding to 50% of the maximum RET efficiency (E(RET)), is a critical parameter for optimization of RET biosensors. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Förster distance. Here, for the first time, we experimentally determine the Förster distance for BRET(1), R(0) = 4.4 nm, and for BRET(2), R(0) = 7.5 nm. The latter is the largest reported value for a genetically encoded RET pair.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Mediciones Luminiscentes/métodos , Transporte de Electrón , Sensibilidad y EspecificidadRESUMEN
In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.
Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Mediciones Luminiscentes/métodos , Péptido Hidrolasas/química , Mapeo de Interacción de Proteínas/métodos , Trombina/química , Sistemas de Computación , Péptido Hidrolasas/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trombina/análisisRESUMEN
Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.
Asunto(s)
Bombyx/anatomía & histología , Bombyx/genética , Regulación de la Expresión Génica , Receptores Odorantes/genética , Órganos de los Sentidos/metabolismo , Caracteres Sexuales , Abdomen , Secuencia de Aminoácidos , Animales , Biología Computacional , Femenino , Genoma de los Insectos/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Masculino , Datos de Secuencia Molecular , FilogeniaRESUMEN
Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein "lipophorin" and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Portadoras/inmunología , Lipoproteínas/inmunología , Mariposas Nocturnas/clasificación , Animales , Antígenos/inmunología , Antígenos/metabolismo , Hemolinfa/inmunología , Ratones , Mariposas Nocturnas/inmunología , Óvulo/inmunología , Factores de TiempoRESUMEN
The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a Kd for racemic JH III of 33 +/- 6 nM. The density of the binding sites is 212 +/- 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [3H]JH III with JH III > JH II > JH I > JH III acid > JH III diol > JHB3 = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells.
Asunto(s)
Proteínas Portadoras/química , Dípteros/química , Hormonas Juveniles/metabolismo , Lipoproteínas , Secuencia de Aminoácidos , Animales , Unión Competitiva , Proteínas Portadoras/metabolismo , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Peso Molecular , Unión ProteicaRESUMEN
A 29-kDa nuclear juvenile hormone (JH)-binding protein from the epidermis of Manduca sexta larvae was purified by using the photoaffinity analog for JH II ([3H]epoxyhomofarnesyldiazoacetate) and partially sequenced. A 1.1-kb cDNA was isolated by using degenerate oligonucleotide primers for PCR based on these sequences. The cDNA encoded a 262-amino acid protein that showed no similarity with other known proteins, except for short stretches of the interphotoreceptor retinoid-binding protein, rhodopsin, and human nuclear protein p68. Recombinant baculovirus containing this cDNA made a 29-kDa protein that was covalently modified by [3H]epoxyhomofarnesyldiazoacetate and specifically bound the natural enantiomer of JH I (Kd = 10.7 nM). This binding was inhibited by the natural JHs but not by methoprene. Immunocytochemical analysis showed localization of this 29-kDa protein to epidermal nuclei. Both mRNA and protein are present during the intermolt periods; during the larval molt, the mRNA disappears but the protein persists. Later when cells become pupally committed, both the mRNA and protein disappear with a transient reappearance near pupal ecdysis. The properties of this protein are consistent with its being the receptor necessary for the antimetamorphic effects of JH.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Insectos , Metamorfosis Biológica/fisiología , Mariposas Nocturnas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , ADN Complementario/aislamiento & purificación , Isomerismo , Hormonas Juveniles/metabolismo , Cinética , Larva , Masculino , Datos de Secuencia Molecular , Peso Molecular , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/fisiología , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Sesquiterpenos/metabolismo , TritioRESUMEN
Retinae of the crab Leptograpsus which had been maintained on a 12-h light/12-h dark cycle were cultured in vitro and exposed to 1 microM okadaic acid (OKA) at 0.75 h before light onset. Control retinae were subjected to the same routine and sampled at the same times without OKA treatment. At the concentration used, OKA totally inhibits types 1 and 2A protein phosphatases, minimally inhibits type 2B, and does not affect type 2C. 1 microM OKA provoked a diminution of rhabdom diameter measured at the level of the photoreceptor nuclei in the dark, some ommatidial cartridges being stripped of rhabdomeral microvilli altogether. After 1-h illumination (225-320 lux), further reduction of rhabdom diameter was modest in control retinae but precipitate in those treated with OKA. After 2 h, control rhabdom diameters showed a further, not significant, decline, but OKA had induced a resynthesis of massive structures with the light-microscopic appearance of rhabdoms. Electron microscopy revealed that they were heterogeneous and of the following kinds: (1) a minority of rhabdoms with normally disposed but distorted microvilli; (2) rhabdoms in the throes of events that parody normal assembly; and (3) rhabdomal volumes occupied by saccular organelles or by pleats or ruffles of irregular architecture. The cytoplasm of all such receptors was packed with free and bound ribosomes and endomembranes. The sequence of events parallels that seen during light-induced degeneration of photoreceptors of the Drosophila mutant w rdgBKS222. Preliminary experiments show that a protein kinase activator SC-9 mimics many of these effects in the dark in the presence of 1 microM OKA. As a working hypothesis, it is proposed that light activates protein kinases via diacylglycerols generated by the phototransduction cascade, and that in both crab retinas challenged with OKA and retinas of rdg BKS222 activation of a nuclear regulatory protein by hyperphosphorylation provokes a runaway transcription whose selectivity and extent remain to be determined.
Asunto(s)
Braquiuros/anatomía & histología , Éteres Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Animales , Drosophila melanogaster/anatomía & histología , Ionóforos/efectos adversos , Luz/efectos adversos , Ácido Ocadaico , Técnicas de Cultivo de Órganos , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/ultraestructura , Retina/citología , Retina/efectos de los fármacos , Degeneración Retiniana/patologíaRESUMEN
Inositol trisphosphate appears to be an excitatory second messenger in the transduction cascade of invertebrate visual photoreceptors. The high time-resolution of visual transduction demands an efficient system for the removal of the second messenger. It is now demonstrated that soluble extracts of crab retina promote rapid magnesium-dependent release of inorganic phosphate from D-myo-1,4,5,-inositol trisphosphate. Experiments in which the inositol trisphosphate had been labelled with 32P in the 4' and 5' positions indicated that both inositol trisphosphatase and bisphosphatase activities are present. The breakdown involves loss of at least one of the pair of vicinal phosphates, which is sufficient to inactivate the compound.
Asunto(s)
Braquiuros/enzimología , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Retina/enzimología , Fosfatos de Azúcar/metabolismo , Animales , Membrana Celular/enzimología , Citosol/enzimología , Inositol 1,4,5-Trifosfato , Inositol Polifosfato 5-Fosfatasas , Magnesio/farmacología , Cloruro de Magnesio , Sistemas de Mensajero SecundarioRESUMEN
The concept of critical period plasticity in rat visual cortex has been studied in terms of changes in the level of filamentous actin and of the 200 kDa neurofilament polypeptide. Our results suggest that the postnatal developmental profile of filamentous actin is affected by visual experience, as a consequence of eye-opening. No such correlation, however, is detected for the 200 kDa neurofilament polypeptide. The significance of these findings in relationship to neuronal plasticity is discussed in terms of changes in the state and equilibrium conditions of the cytoskeletal proteins.