Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk.

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.

Classifying continuous, real-time e-nose sensor data using a bio-inspired spiking network modelled on the insect olfactory system.

In many application domains, conventional e-noses are frequently outperformed in both speed and accuracy by their biological counterparts. Exploring potential bio-inspired improvements, we note a number of neuronal network models have demonstrated some success in classifying static datasets by abstracting the insect olfactory system. However, these designs remain largely unproven in practical settings, where sensor data is real-time, continuous, potentially noisy, lacks a precise onset signal and accurate classification requires the inclusion of temporal aspects into the feature set. This investigation therefore seeks to inform and develop the potential and suitability of biomimetic classifiers for use with typical real-world sensor data. Taking a generic classifier design inspired by the inhibition and competition in the insect antennal lobe, we apply it to identifying 20 individual chemical odours from the timeseries of responses of metal oxide sensors. We show that four out of twelve available sensors and the first 30 s (10%) of the sensors' continuous response are sufficient to deliver 92% accurate classification without access to an odour onset signal. In contrast to previous approaches, once training is complete, sensor signals can be fed continuously into the classifier without requiring discretization. We conclude that for continuous data there may be a conceptual advantage in using spiking networks, in particular where time is an essential component of computation. Classification was achieved in real time using a GPU-accelerated spiking neural network simulator developed in our group.

Experimental determination of the Förster distance for two commonly used bioluminescent resonance energy transfer pairs.

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.

Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.

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.

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori.

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.

Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae).

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.

Comparison of efficacy and ease of handling of salmeterol and terbutaline powder inhalers.

A multicentre, randomised, open, parallel-group study was performed to compare the efficacy, tolerability and ease of handling of salmeterol xinafoate 50 micrograms twice daily via the Diskus inhaler with terbutaline sulphate 500 micrograms four times daily via the Turbuhaler inhaler. Two hundred and sixty-three patients (aged 18-79 years, baseline FEV1 50-90% predicted, mean PEFR 85% of response to salbutamol) were randomised to treatment with salmeterol (n = 136) or terbutaline (n = 127). A statistically significant difference in favour of salmeterol was seen between treatment groups for the primary efficacy variable, mean morning PEFR (difference in adjusted means 25.4 l/min, p < 0.001). Within the groups randomised to each device, ease of handling assessments favoured the Diskus inhaler over the Turbuhaler inhaler. More patients liked the Diskus inhaler than the Turbuhaler inhaler (98% vs 72%, p < 0.001). The Diskus inhaler received better scores than the Turbuhaler inhaler for all features assessed in the device questionnaire.

Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.

Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina.

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.

A nuclear juvenile hormone-binding protein from larvae of Manduca sexta: a putative receptor for the metamorphic action of juvenile hormone.

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.

Investigation of numbers and motility of spermatozoa in reproductively active and socially suppressed males of two eusocial African mole-rats, the naked mole-rat (Heterocephalus glaber) and the Damaraland mole-rat (Cryptomys damarensis).

Reproductive tracts and spermatozoa from reproductively active and reproductively suppressed non-breeding males from two species of eusocial African mole-rats Cryptomys damarensis and Heterocephalus glaber were examined. In two captive colonies of Heterocephalus glaber, reproductive tracts from seven non-breeding males removed from their colonies, and housed singly for 5-6 weeks to cause reproductive activation, were compared with reproductive tracts from seven non-breeding males. The body weight of the separated, reproductively active males increased significantly (P < 0.01), and the mean testis weights relative to body weight of the reproductively active males were significantly larger (P < 0.05) than those of non-breeding males. The number of spermatozoa, in one half of the reproductive tract, was higher in active males than in non-breeding males (mean +/- SEM: 8.59 x 10(6) +/- 2.69 x 10(6) versus 1.78 x 10(6) +/- 1.43 x 10(6), respectively; P < 0.05). In addition, six of the seven reproductively active males, but only two of seven non-breeding males, had motile spermatozoa. A total of 28 wild Cryptomys damarensis from two colonies were examined in the field. The testis weights relative to body weight of breeding males (n = 7) were higher than those of non-breeding males (n = 19; P < 0.01), but the number of spermatozoa did not differ significantly between the two groups (0.13 x 10(6) +/- 0.06 x 10(6), n = 7 versus 0.29 x 10(6) +/- 0.14 x 10(6), n = 21, respectively). Breeding and non-breeding males produced similar numbers of motile spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of retinal degeneration in a crab by light and okadaic acid in vitro: comparison with the Drosophila light-dependent retinal degeneration mutant w rdgBKS222.

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.

Inositol trisphosphatase and bisphosphatase activities in the retina of crab.

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.

Postnatal developmental profiles of filamentous actin and of 200 kDa neurofilament polypeptide in the visual cortex of light- and dark-reared rats and their relationship to critical period plasticity.

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.