The expression pattern of four odorant-binding proteins in male and female silk moths, Bombyx mori.

Four recombinant odorant-binding proteins of Bombyx mori, pheromone-binding protein (PBP), general odorant-binding protein 1 (GOBP1), general odorant-binding protein 2 (GOBP2) and antennal binding protein X (ABPX), were expressed in E. coli and used to raise polyclonal antisera. Immunoblots of antennal homogenates showed that these antisera were specific. In Western blot analysis and immunocytochemical labelling experiments, the sera against recombinant PBP and GOBP2 of B. mori gave identical results as sera against native PBP and GOBP2 of Antheraea polyphemus, respectively, thus confirming earlier results obtained with the latter. Labelling consecutive cross sections of various sensillum types with all four antisera revealed different labelling patterns in male and female sensilla (s.) trichodea and s. basiconica. Long s. trichodea in males and females represented uniform labelling types, whereas for short s. trichodea, s. intermedia, and s. basiconica a great variety of labelling patterns was observed, some being more common than others. Long s. trichodea, which in males are uniformly tuned to the pheromone components bombykol and bombykal, all strongly expressed PBP; labelling with antisera against the other three odorant-binding proteins hardly was above background, only in some hairs GOBP1 was expressed somewhat more strongly. Long s. trichodea of females, which respond specifically to linalool and benzoic acid, showed a different labelling pattern. Here, we observed strong labelling with antibodies against GOBP2 and medium labelling with anti-GOBP1, sometimes with anti-ABPX. S. basiconica in both sexes most commonly co-expressed GOBP1 and GOBP2, but other patterns were occasionally found, with some of them showing PBP expression, also in females. The great variety of labelling types in short s. trichodea, s. intermedia, and s. basiconica suggests a similar variety of functional subtypes as observed in plant odour-sensitive sensilla of other moth species.

Ligand binding to six recombinant pheromone-binding proteins of Antheraea polyphemus and Antheraea pernyi.

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, ( E, Z)-6,11-hexadecadienyl acetate ((3)H-Ac1) and ( E, Z)-6,11-hexadecadienal ((3)H-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone ((3)H-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of (3)H-Ac1. In competition experiments with (3)H-Ac1 and the third unlabelled pheromone component, ( E, Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound (3)H-Ald only. All of the six PBPs strongly bound (3)H-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for (3)H-Ac1 and (3)H-Ald bound to PBPs. No pH influence was found for (3)H-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1-3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation.

Immunolocalization of odorant-binding proteins in noctuid moths (Insecta, Lepidoptera).

Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.

Female Attacus atlas respond to pheromones of Antheraea polyphemus: a comparative electrophysiological and biochemical study.

Female Attacus atlas respond electrophysiologically to both of the Antheraea polyphemus pheromone components (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-6,11-hexadecadienal. Moreover, they possess a pheromone-binding protein (PBP) and general odorant-binding proteins (GOBPs), as well as a pheromone-degrading sensillar esterase and aldehyde oxidase enzymes. They show no electroantennogram responses to their own gland extract. In contrast, female A. polyphemus do not respond to their own or to A. atlas pheromone. Male A. atlas do not detect any of the A. polyphemus compounds but only the conspecific female gland extracts. Both male A. atlas and female A. polyphemus possess PBP and GOBP but lack the pheromone-degrading esterases of male Antheraea. The results indicate that the two species use quite distinct classes of chemicals as pheromones. In spite of this, the N-terminal amino acid sequences of the PBPs show homology of 68%.

Chemosensory proteins from the proboscis of mamestra brassicae.

Soluble, low molecular weight proteins were immunodetected in proboscis extracts of Mamestra brassicae males by Western blot, using antibodies raised against the general odorant-binding protein of the moth Antheraea polyphemus. The same antibodies weakly labelled the sensillum lymph and subcuticular space of sensilla styloconica on ultrathin sections of the proboscis. The morphology of sensilla styloconica is described. The immunodetected proteins yielded several N-terminal sequences, three of which showed strong affinity for tritiated analogues of pheromonal compounds of M. brassicae in binding assays. The cDNAs coding for these sequences were cloned and it was shown that the new proteins are related to the OS-D protein of Drosophila. They are named chemosensory proteins (CSP-MBRA:A1-CSP-MBRA:A5 and CSP-MBRA:B1 and CSP-MBRA:B2) and may have an odorant-binding protein-like function. A common localization in both olfaction and taste organs suggests a physiological role depending on the cellular environment.

Identification of PLC beta and PKC in pheromone receptor neurons of Antheraea polyphemus.

Two proteins of the IP3 transduction pathway were identified by Western blots in homogenates of isolated pheromone-sensitive sensilla of the silkmoth Antheraea polyphemus. A 110 kDa protein was recognized by an antiserum raised against the Drosophila phospholipase C beta (PLC beta p121) and a 80kDa protein was labelled by an antiserum against a synthetic peptide of a conserved region of protein kinase C (PKC). Incubation of homogenized sensory hairs with the main sex pheromone component, (E,Z) 6-11 hexadecadienyl acetate, resulted in a 6-fold increase in the activity of PKC compared to controls without pheromone. In contrast, incubation with pheromone did not affect the activity of protein kinase A (PKA). Activation of PKC by the membrane permeable dioctanoylglycerol led to excitation of the pheromone-sensitive receptor neurons. These data support the current concept that pheromone perception of moths is mediated by the IP3 transduction pathway.

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.

Effectiveness of clopidogrel and aspirin versus ticlopidine and aspirin in preventing stent thrombosis after coronary stent implantation.

BACKGROUND: Ticlopidine has been shown to reduce the incidence of stent thrombosis compared with warfarin, but it may cause serious hematological side effects. Clopidogrel, a new thienopyridine derivative, may be a safe alternative to ticlopidine. The aim of this study was to compare the safety and efficacy of clopidogrel and aspirin with those of ticlopidine and aspirin in patients undergoing coronary stent implantation. METHODS AND RESULTS: The population of this study consisted of 2 groups: patients who underwent coronary stenting and were treated with ticlopidine and aspirin (TA group, n=1406), and patients who underwent coronary stenting followed by treatment with clopidogrel and aspirin (CA group, n=283). At 1-month follow-up, there was no difference in stent thrombosis (1.5% versus 1.4%, P=1.0) or major adverse cardiac events (3.1% versus 2.4%, P=0. 85) between the TA and CA groups, respectively. The probability of any side effect (neutropenia, diarrhea, rash) was significantly higher in the TA group (10.6% versus 5.3%, P=0.006; relative risk, 0. 53; CI, 0.32 to 0.86). CONCLUSIONS: These data suggest that clopidogrel may be an effective pharmacological regimen after coronary stent implantation. Furthermore, the simpler dosing regimen, the absence of neutropenia, and the lower frequency of other side effects make it a safe alternative to ticlopidine.

Heterogeneity of odorant-binding proteins in the antennae of Bombyx mori.

Different odorant-binding proteins (OBPs) were isolated from total antennal homogenates of male and female Bombyx mori. Proteins were separated according to their isoelectric point by using preparative fast-flow isoelectrofocusing. Odorant-binding proteins were identified in immunoblots by antisera raised against the pheromone-binding protein (anti-PBP) and the general odorant-binding protein (anti-GOBP2) of Antheraea polyphemus. Four proteins cross-reacting with anti-PBP were detected in males and two in females, while three proteins cross-reacting with anti-GOBP2 were found in males and five in females. Both anti-PBP and anti-GOBP2 cross-reacting proteins had an apparent molecular weight of 15-16 kDa. In parallel, the same two antisera were used in immunocytochemical studies in order to determine the distribution of these proteins within the various subtypes of olfactory sensilla. The presence of multiple odorant-binding proteins within one moth species as well as their complex distribution pattern support the suggestion that soluble OBPs might have a function in odorant discrimination.

G-protein activation, identification and immunolocalization in pheromone-sensitive sensilla trichodea of moths.

Electrophysiological in situ recordings from pheromone-sensitive sensilla trichodea of Bombyx mori males with a recording pipette which contained G-protein-activating fluoride, showed receptor cell activity similar to that evoked by pheromone stimulation. This suggests that G-proteins might be physiologically active in olfactory sensilla of insects in situ. Biochemical experiments using specific antibodies revealed the presence of G-protein, belonging to the Gq family, in antennal preparations. Similar G-protein was identified in sensory hair preparations of Antheraea pernyi which contained only cuticle, sensillum lymph and dendritic material. Moreover, the absence of this G-protein in pure sensillum lymph preparations indicates its association with the receptive dendrites. This particular association could be shown by immunolabelling studies at the ultrastructural level. Strong specific labelling of membranes of receptor-cell dendrites was found in all types of olfactory sensilla present on the antenna of the silkmoths. Additional specific labelling of apical membranes of auxiliary cells, epidermal cells and membranes forming the axon/glia interface demonstrated that this G-protein is not restricted to the sensory dendrites and that other signal-transduction pathways could be present at these membranes. In summary, the experiments imply a participation of G-protein of the Gq family in signal transduction of olfactory receptor cells in moths.

Identification of cytoskeletal proteins in the antennae of the silkmoths Antheraea polyphemus and A. pernyi.

Although the overall cytoskeletal morphology of the olfactory dendrite in the antennae of the silkmoths Antheraea polyphemus and A. pernyi is known, the cytoskeleton proteins that structurally and functionally support these structures remain to be identified in this paper, we describe the identification of tubulin, actin and intermediate filament-like proteins in the olfactory dendrites, and motor proteins such as kinesin and unconventional myosin in the antennal branches by the use of antibodies. We also show that the tubulins within the olfactory dendrites and in the antennal branches are acetylated. This study provides valuable information concerning the possible role of these proteins in transduction, transport and motility, as is evident in other systems.

Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea).

A 19 kDa protein has been purified by gel filtration and anion-exchange chromatography from the antennae of Carausius morosus. Its amino terminal amino acid sequence shows significant similarity (30% identity) with another putative odorant-binding protein, the so called OS-D protein isolated from the antennae of Drosophila melanogaster; only 20% of its amino acids are shared with some members of Lepidoptera pheromone-binding proteins. Polyclonal antibodies, raised against a synthetic amino terminal peptide cross-react with 19 kDa band in the legs extracts, but not with soluble proteins from other parts of the body. The amino terminal sequence of this protein, purified from the legs was identical with that of the antennal protein.

Odorant-binding proteins in insects.

This paper reviews the characteristics of pheromone and odorant-binding proteins (OBP) in insects, with particular reference to Lepidoptera. They are small (15 kDa) soluble proteins, very concentrated in the lymph of chemosensory sensilla and belonging to two major classes, pheromone-binding proteins (PBP) and general odorant-binding proteins. They represent the insect equivalent of vertebrate OBP. The main unsolved question with OBP of insects and vertebrates regards their physiological role in olfactory transduction. The recent discovery of several types of OBP in the same animal species suggests that these proteins may be involved in the discrimination of odours.

Esterase activity in the olfactory sensilla of the silkmoth Antheraea polyphemus.

We studied in individual males of Antheraea polyphemus the activity of the sensillar esterase, a pheromone-degrading enzyme present in the sensillum lymph surrounding the olfactory receptor cells. In parallel, receptor potentials from single pheromone-sensitive sensilla trichodea were recorded. Our screening revealed a large variability of the enzyme activity in individuals with similar electrophysiological responses. In some moths the sensillar esterase was not detectable, i.e. present with 100-fold less activity. However, such variable esterase activity showed no correlation to the time course of the receptor potential. Thus, enzymatic pheromone degradation does not seem to be involved in the rapid pheromone inactivation at the end of the stimulus, but rather serves as the final pheromone sequestration step.

[Physiological functions of odorant-binding proteins]. / Fiziologicheskie funktsii odorant-sviazyvaiushchikh belkov.

The chemical characteristics of odorant-binding proteins (OBPs) are summarised and compared with those of other types of binding proteins involved in chemical communication. The recent finding of multiple forms of OBPs in the same animal species could suggest an odour discriminating role for these proteins. In this respect, the vertebrates' olfactory system is similar to other chemoreception systems, such as those present in insects for detecting pheromones and general odours, bacterial chemotaxis and taste.