Synthetic peptide mimicking of binding sites on olfactory receptor protein for use in 'electronic nose'

A putative tertiary structure model of the dog's olfactory receptor (olfd canfa) is established in this study. By using a target odorous compound (trimethylamine), it is possible to locate the most plausible binding sites between the receptor model structure and the target odorous molecules through computer docking simulations. The two short oligo-peptide sequences (orp61 and orp188) for trimethylamine sensing were identified, synthesized, purified and coated onto the surface of the separate piezoelectric gold electrodes. These two peptides show a high binding capability for trimethylamine. To further enhance the sensitivity of the polypeptides towards the target compound, the polarity and the degree of docking were changed by a site-specific modification technique. The orp61 sequence was modified by substituting two amino acids in the binding pocket resulting in 33% increase in sensitivity towards trimethylamine and reduced noises from other non-target chemicals. The techniques used in the present study offer a unique approach for synthesizing peptides in mimicking binding domain of olfactory receptors. The approach can be easily applied to further development of recognized molecules for gas sensing, especially for use in 'electronic noses'.     

Olfactory cell-based biosensor: a first step towards a neurochip of bioelectronic nose

Human olfactory system can distinguish thousands of odors. In order to realize the biomimetic design of electronic nose on the principle of mammalian olfactory system, this article reports an olfactory cell-based biosensor as a real bionic technique for odorants detection. Effective cultures of olfactory receptor neurons and olfactory bulb cells have been achieved on the semiconductor chip. Using light-addressable potentiometric sensor (LAPS) as sensing chip to monitor extracellular potential of the neurons, the response under stimulations of the odorants or neurotransmitters, such as acetic acid and glutamic acid, was tested. The results demonstrate that this kind of hybrid system of LAPS and olfactory neurons, which is sensitive to odorous changes, has great potential and is promising to be used as a novel neurochip of bioelectronic nose for detecting odors.     

Development of a fully integrated microdistillation flow injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system comprised a fully integrated microdistillation flow injection (MDFI) system for low level ammonia analysis was reported. In this system, the microdistillation separation step was incorporated into the flow injection manifold and the ammonia gas sensing probe sensing element was replaced by a flow-through micro-pH electrode which sensed the change in pH of a flowing collector solution caused by the dissolution of distilled ammonia gas, in a process analogous to that occurring in the internal solution of the gas sensing probe. A computerised control and data acquisition system was constructed for this system using a commercially available data acquisition card which offered many advantages such as improved data acquisition rates and control over the system components, as well as good graphics display and data processing options. The system was optimised using a multi-variable simplex optimisation technique.     

The role of frass and cocoon volatiles in host location by Monodontomerus aeneus, a parasitoid of Megachilid solitary bees

Monodontomerus aeneus (Fonscolombe) is a parasitic wasp that oviposits on the prepupae and pupae of Osmia cornuta (Latreille) and other solitary bee species. A two-armed olfactometer was used to test the olfactory attractiveness of O. cornuta prepupae, cocoon, and larval frass to female M. aeneus. Both cocoon and frass attracted the female parasitoids, but frass alone was more attractive than the cocoon and the cocoon with frass was more attractive than frass alone. Female parasitoids were not attracted by the host prepupa. M33 (methanol) was the organic volatile most emitted by cocoons and m61 (acetic acid) was the compound most emitted by frass. However, cocoons showed higher emission for almost all compounds, including m61 (acetic acid). Although acetic acid alone attracted M. aeneus, a complex volatile signal is probably involved in the attraction process because the ratio of acetic acid and acetaldehyde characteristic of the frass was more attractive than other ratios.     

Changes in the serotonergic system in the main olfactory bulb of rats unilaterally deprived from birth to adulthood

The serotonergic system plays a key role in the modulation of olfactory processing. The present study examined the plastic response of this centrifugal system after unilateral naris occlusion, analysing both serotonergic afferents and receptors in the main olfactory bulb. After 60 days of sensory deprivation, the serotonergic system exhibited adaptive changes. Olfactory deprivation caused a general increase in the number of fibres immunopositive for serotonin but not of those immunopositive for the serotonin transporter. HPLC data revealed an increase in serotonin levels but not in those of its major metabolite, 5-hydroxyindole acetic acid, resulting in a decrease in the 5-hydroxyindole acetic acid/serotonin ratio. These changes were observed not only in the deprived but also in the contralateral olfactory bulb. Double serotonin-tyrosine hydroxylase immunolabelling revealed that the glomerular regions of the deprived olfactory bulb with a high serotonergic fibre density showed a strong reduction in tyrosine hydroxylase. Finally, the serotonin(2A) receptor distribution density and the number of juxtaglomerular cells immunopositive for serotonin(2A) receptor remained unaltered after olfactory deprivation. Environmental stimulation modulated the serotonergic afferents to the olfactory bulb. Our results indicate the presence of a bilateral accumulation of serotonin in the serotonergic axon network, with no changes in serotonin(2A) receptor density after unilateral olfactory deprivation.     

Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

BTEX-Contaminated Gas Treatment in a Shallow, Sparged, Suspended-Growth Bioreactor

Treatment of a gas contaminated with a mixture of benzene, toluene, ethylbenzene, and o-xylene (BTEX) compounds in a 40-cm-deep laboratory-scale bioreactor containing suspended biomass was investigated. Gas treatment efficiency was not significantly impacted by different BTEX mixtures, and approximately 99% removal was achieved for volumetric loadings of 11 to 18 mg-BTEX/L-reactor volume/hr (specific biomass loadings of 0.27 to 0.83 g-BTEX/g-VSS/d; inlet concentrations of total BTEX of 2.3 to 4.3 mg/L) and operational solids retention times (SRTs) of 1.7, 2.7, and 9.2 days. Maximum specific biodegradation rates of the reactor biomass increased as the reactor SRTs decreased. Under specific loadings greater than 1 g-BTEX/g-VSS/d the gas treatment became biokinetically limited, such that BTEX and unidentified BTEX metabolites accumulated in the bioreactor liquid over time. BTEX gas-liquid mass transfer was sufficient in the 40-cm-deep sparged liquid reactor to provide high BTEX treatment efficiency.     

Chemical Cleavage of Bovine β-Lactoglobulin by BNPS-Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine β-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): identification of two Asp-Gly-Arg-containing domains involved in the mitogenic activity of bFGF in endothelial cells.

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38-61) and bFGF (82-101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bFGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2-diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp-Gly-Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR- and RGD-containing tetra- and heptapeptides inhibit the mitogenic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR-and RGD-containing peptides inhibit the binding of 125I-bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38-61 and 82-101, both containing a core DGR sequence, represent two "activation" domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.     

Antigenic structure of histone H1°

In order to study the antigenic structure of histone Hl° the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1° antiserum. The C-terminal fragments 99–193 (obtained following acetic acid hydrolysis) and 107–193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1–30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1–22 and 1–28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1° are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.     

Trimethylamine modulates dauer formation,neurodegeneration, and lifespan through tyra‐3/daf‐11 signaling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal''s major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA‐3, a conserved G protein‐coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF‐11 signaling through TYRA‐3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food‐sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson''s disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems.     

A peptide receptor-based bioelectronic nose for the real-time determination of seafood quality

We herein report a peptide receptor-based bioelectronic nose (PRBN) that can determine the quality of seafood in real-time through measuring the amount of trimethylamine (TMA) generated from spoiled seafood. The PRBN was developed using single walled-carbon nanotube field-effect transistors (SWNT-FETs) functionalized with olfactory receptor-derived peptides (ORPs) which can recognize TMA and it allowed us to sensitively and selectively detect TMA in real-time at concentrations as low as 10fM. Utilizing these properties, we were able to not only determine the quality of three kinds of seafood (oyster, shrimp, and lobster), but were also able to distinguish spoiled seafood from other types of spoiled foods without any pretreatment processes. Especially, the use of small synthetic peptide rather than the whole protein allowed PRBNs to be simply manufactured through a single-step process and to be reused with high reproducibility due to no requirement of lipid bilayers. Furthermore, the PRBN was produced on a portable scale making it effectively useful for the food industry where the on-site measurement of seafood quality is required.     

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

Identification of cytochromes o and a3 as functional terminal oxidases in the thermophilic bacterium, PS3

Low-temperature photodissociation spectra of membranes from the thermophile PS3 reveal cytochromes o and a₃. The latter reacts with O₂ at −103°C to give a light-insensitive compound(s), but the initial stages of O₂ binding to cytochrome o could not be studied under these conditions. Photochemical action spectra identify cytochromes a₃ and o, but not a CO-binding c-type cytochrome, as functional terminal oxidases in this bacterium.     

Electron microscopic observation of mu-opioid receptor in the rat area postrema.

A simple preembedding avidin-biotin-peroxidase complex technique was used to study the ultrastructural localization of mu-opioid receptor in the rat area postrema. By using low concentrations of the first antiserum for incubation with a short reaction time to 3,3'-diaminobenzidine, the immunostaining was faint at the light microscopic level. However, at the electron microscopic level, strong immunoreaction was observed. Mu-Opioid receptors were found to be localized on the postsynaptic membrane of dendrites, extrasynaptic plasma membrane, and the surface of the small, clear vesicles in axon terminals. Of the total 283 immunopositive profiles observed, 68.2% (193 of 283) were dendrites, 29.3% (83 of 283) were axon terminals, and 2.5% (7 of 283) were myelinated axons. No immunostained neuron bodies were found in the present study; 109 mu-opioid receptor immunoreactive dendrites received synapses (56.5%, 109 of 193) from nonimmunoreactive (84.4%, 92 of 109) or immunoreactive (15.6%, 17 of 109) axon terminals, whereas 84 dendrites (43.5%, 84 of 193) were found without receiving synapses. The present study shows that the mu-opioid receptor in the area postrema plays a role mainly at the synapses.     

The amino acid sequence of the enterotoxin from Clostridium perfringens type A

The amino acid sequence of the enterotoxin from Clostridium perfringens type A was determined by analysis of peptides derived from the protein by digestion with trypsin chymotrypsin, thermolysin, pepsin, a lysine-specific protease. S. aureus V8 protease and a proline-specific protease, and fragments generated by cleavage with cyanogen bromide or by dilute acetic acid in 7 M guanidine HCl. The sequence which is complete except for the definite order of 3 small peptides between residues 88 and 103 consists of 309 amino acids and contains a correction to our preliminary announcement [(1984) FEMS Symp. 24, 329-330].     

Development of an automated flow-injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system for low level ammonia analysis is examined. The system features an on-line microdistillation preconcentration unit, which was used as an on-line sample pretreatment step in an ammonia gas-sensing probe flow injection system. A simple, low cost computerised control and data acquisition system was designed using a commercial pH meter with RS-232 interface and in-house designed control system. The system offered a practical and effective means of extending the detection limit of commercial available ammonia gas sensing probes to 5 μg/1 NH₃N.     

Identification of vertebrate volatiles stimulating olfactory receptors on tarsus I of the tick Amblyomma variegatum Fabricius (Ixodidae)

Bovine odour excites olfactory receptor(s) in a wall-pore olfactory sensillum on the anterior pit of Haller's organ in Amblyomma variegatum. Gas chromatography-coupled electrophysiology recordings from this sensillum reveal the presence of 4 active compounds in bovine odour. The two strongest stimulants were identified as 2-nitrophenol and 4-methyl-2-nitrophenol by gas chromatography-coupled mass spectrometry, and by matching electrophysiological activity of synthetic analogues. Synthetic analogues of known vertebrate-associated volatiles also stimulate other olfactory receptors in sensilla on the surface of tarsus I: a lactone receptor responding to -valerolactone and 6-caprolactone; different fatty acid receptor types responding best to either pentanoic acid, 2-methylpropanoic acid or to butanoic acid; three receptors responding to NH₃; and one receptor responding to 3-pentanone. Gas chromatography-coupled mass spectrometry analysis of vertebrate volatiles revealed presence of a number of these olfactory stimulants in concentrates of rabbit and steer odour, i.e. 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, and -valerolactone.Abbreviations GC-EL gas chromatography-coupled electrophysiological recording - GC-MS gas chromatography-coupled mass spectrometry     

Cloning and expression of guanylin. Its existence in various mammalian tissues.

Guanylin (PNTCEICAYAACTGC) is a peptide recently isolated from the intestine, the actions of which appear to be mimicked by bacterial heat-stable enterotoxins (Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and Smith, C. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 947-951). A cDNA clone encoding the peptide was isolated from a rat intestinal cDNA library using a degenerate oligonucleotide probe. The mRNA (approximately 0.8-0.9 kilobase) encoding the peptide contained an open reading frame of 115 amino acids, including an amino-terminal signal peptide. The carboxyl-terminal region of the predicted polypeptide contained a sequence identical to guanylin, but the 15-amino acid peptide likely represents an artifact of previous acetic acid extraction methods, since an aspartate residue precedes the amino-terminal proline. A lysine-lysine dipeptide bond is one likely processing site of pro-guanylin and would generate a 60-amino acid mature peptide. Other potential cleavage sites exist at single lysine and arginine residues, which could result in peptides ranging from 22 to 56 amino acids. Transfection of COS-7 cells with the guanylin cDNA resulted in the expression of a secreted protein of M(r) 10,000. The expressed proguanylin failed to elevate cyclic GMP concentrations in human colonic T84 cells, but acetic acid treatment of pro-guanylin activated it and resulted in large elevations of cyclic GMP. Guanylin mRNA was prevalent in rat intestine but was also found in low abundance in adrenal gland, kidney, and uterus/oviduct. Guanylyl cyclase C, the apparent guanylin receptor, was found in abundant amounts in the intestine by Northern analysis, and by the polymerase chain reaction or cDNA cloning it was also found in adrenal gland, airway epithelial cells, brain, and olfactory and tracheal mucosa. Therefore, the ligand and apparent receptor (guanylyl cyclase C) both originate from mammalian genes, and are expressed in various mammalian tissues.     

Ultracytochemical localization of particulate guanylate cyclase after stimulation with natriuretic peptides in lamb olfactory mucosa

Summary The ultracytochemical localization of particulate guanylate cyclase has been studied in lamb olfactory mucosa after activation with rat atrial natriuretic factor (rANF), porcine brain natriuretic peptide (pBNP), porcine C-type natriuretic peptide (pCNP) or rat brain natriuretic peptide (rBNP). Particulate guanylate cyclase is the receptor for these peptides and recently two subtypes of the cyclase have been identified. These isoforms are stimulated differently by ANF, BNP and CNP. Under our experimental conditions, rANF, pCNP and pBNP were strong activators of particulate guanylate cyclase in lamb olfactory mucosa, as demonstrated by the presence of reaction product. Samples incubated in basal conditions without rANF, pCNP or pBNP, or samples incubated in presence of rBNP did not reveal any cyclase activity. The rANF-stimulated cyclase activity was localized in the apical portion of olfactory epithelium. pCNP-stimulated guanylate cyclase was detected to the lamina propria in association with secretory cells of Bowman's glands and with cells in close relation with Bowman's glands (elongated cells and myoepithelial cells). The cyclase activity stimulated by pBNP was limited to cells of Bowman's glands. The present data indicate that ANF and CNP are recognized by different receptors and that BNP and CNP bind to the same receptor.