Protein kinase C stimulates PtdIns-4,5-P2-phospholipase C activity.
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The tumour promoter, phorbol ester 12,13-dibutyrate (PDBu), acts on rectal palisadic epithelial cells and mimics the effects of neuroparsin, an antidiuretic neuronal hormone isolated from nervous lobes of the African locust corpora cardiaca. PDBu stimulated Ca2+-dependent phospholipase C (PLC) activity resulting in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production, increased cytosolic free calcium (monitored with the probe indo-1) and rectal fluid resorption. A 15-min pre-treatment with polymyxin B (PMXB), a protein kinase C (PKC) inhibitor acting at the phosphatidylserine (PS) binding site, suppressed PDBu stimulatory effects on free calcium entry and fluid resorption but not on phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) breakdown. On the contrary, bisindolylmaleimide Ro 32-0432 (which inhibits PKC at its ATP binding site) abolished entirely PDBu-stimulated PLC activity. It was concluded that two PKC are involved in transduction of the antidiuretic signal of neuroparsin. One PKC is PMXB sensitive and stimulates biological response after cytosolic free Ca2+ increase, while another PKC, insensitive to the PKC inhibitor, regulates the processes induced by the former PKC. Since PMXB-insensitive PKC exerts a stimulatory effect on PtdIns-4,5-P2-PLC production, this original mechanism may be considered as a new signalling pathway under control of PKC. (+info)
The vibrational startle response of the desert locust Schistocerca gregaria.
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Substratum vibrations elicit a fast startle response in unrestrained quiescent desert locusts (Schistocerca gregaria). The response is graded with stimulus intensity and consists of a small, rapid but conspicuous movement of the legs and body, but it does not result in any positional change of the animal. With stimuli just above threshold, it begins with a fast twitch of the hindlegs generated by movements of the coxa-trochanter and femur-tibia joints. With increasing stimulus intensity, a rapid movement of all legs may follow, resulting in an up-down movement of the whole body. The magnitude of both the hindleg movement and electromyographic recordings from hindleg extensor and flexor tibiae muscles increases with stimulus amplitude and reaches a plateau at vibration accelerations above 20 m s(-)(2) (peak-to-peak). Hindleg extensor and flexor tibiae muscles in unrestrained animals are co-activated with a mean latency of 30 ms. Behavioural thresholds are as low as 0. 47 m s(-)(2) (peak-to-peak) at frequencies below 100 Hz but rise steeply above 200 Hz. The response habituates rapidly, and inter-stimulus intervals of 2 min or more are necessary to evoke maximal reactions. Intracellular recordings in fixed (upside-down) locusts also revealed co-activation of both flexor and extensor motor neurones with latencies of approximately 25 ms. This shows that the neuronal network underlying the startle movement is functional in a restrained preparation and can therefore be studied in great detail at the level of identified neurones. (+info)
Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria.
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Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40-50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29-Cys38 and Cys57-Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera. (+info)
Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III.
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Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic alpha-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III. (+info)
Phosphorylated proteins are involved in sister-chromatid arm cohesion during meiosis I.
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Sister-chromatid arm cohesion is lost during the metaphase I/anaphase I transition to allow homologue separation. To obtain needed information on this process we have analysed in grasshopper bivalents the sequential release of arm cohesion in relation to the behaviour of chromatid axes. Results show that sister axes are associated during early metaphase I but separate during late metaphase I leading to a concomitant change of chromosome structure that implies the loss of sister-kinetochore cohesion. Afterwards, homologues initiate their separation asynchronously depending on their size, and number and position of chiasmata. In all bivalents thin chromatin strands at the telomeres appeared as the last point of contact between sister chromatids. Additionally, we have analysed the participation of phosphoproteins recognised by the MPM-2 monoclonal antibody against mitotic phosphoproteins in arm cohesion in bivalents and two different kinds of univalents. Results show the absence of MPM-2 phosphoproteins at the interchromatid domain in mitotic chromosomes and meiotic univalents, but their presence in metaphase I bivalents. These phosphoproteins are lost at the onset of anaphase I. Taken together, these data have prompted us to propose a 'working' model for the release of arm cohesion during meiosis I. The model suggests that MPM-2 phosphoproteins may act as cohesive proteins associating sister axes. Their modification, once all bivalents are correctly aligned at the metaphase plate, would trigger a change of chromosome structure and the sequential release of sister-kinetochore, arm, and telomere cohesions. (+info)
Spatiotemporal structure of olfactory inputs to the mushroom bodies.
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A requirement to understand mushroom body (MB) function is to characterize the operations (or transformations) that they impose on incoming signals. Understanding the nature of these integrative operations requires an understanding of the inputs from other brain areas. By inputs we mean not only the anatomical pathways leading to the MBs, but also the dynamic structure of the inflow of sensory (and other) signals. Neurons are complex, capacitative, and generally nonlinear devices that transform barrages of neurochemical packets into electrical waveforms. Their modes of operation are intrinsically time dependent and therefore, their functions or roles in a circuit cannot be inferred only from structural data. Thanks to elegant anatomical, behavioral, genetic, and molecular (for review, see Crittenden et al. 1998; Hammer and Menzel 1998; Heisenberg 1998; Wolf et al. 1998) studies, there is convincing evidence that MB circuits are involved, at least in fruit flies and honeybees, in some forms of odor integration and learning. In vivo electrophysiological studies of MB neurons, however, are rare and mainly restricted to individual (or small populations of) so-called extrinsic neurons, that is, those whose processes link MBs with other brain areas (Schildberger 1983, 1984; Homberg 1984; Hammer 1993; Mauelshagen 1993; Li and Strausfeld 1997). Kaulen et al. (1984) examined extracellular potentials in the MBs of bees, using current source density analysis, and more recently, Laurent and Naraghi (1994) provided a description of stimulus-evoked activity in Kenyon cells (KCs), the intrinsic neurons of the MBs, using intracellular recordings. In this short review we will summarize the recent results from our laboratory in an attempt to provide a description of the spatiotemporal structure of olfactory inputs to the MBs and their intrinsic neurons. We will focus only on the encoding of odor quality. We will then speculate on the possible role of MB circuits for olfactory processing. (+info)
Neurons provide critical signals that regulate both the number and differentiation of glia. In addition, glia are attracted to and enwrap neuronal axonal processes. FGF-like signalling is thought to be one of the many potential axon-derived morphogenetic signals, however, the multiple roles of FGFs have made experimental tests of these signals difficult in vivo. In the Drosophila FGF receptor mutant heartless, glia migrate to axons, but fail to elongate around them. This study shows that in the similar but larger grasshopper CNS, FGF signalling is likely to mediate one step in the close interaction between glia and axons. FGF2-coated beads attract glia in the CNS and compete with axons for their resident, enwrapped glia. In addition, bath applied FGF2 causes mature axonal glia, which normally enwrap axon tracts, to round up. FGF2 activates the product of the grasshopper heartless FGF receptor gene and probably interferes with the normal function of an endogenous axon-associated FGF-like molecule. It is proposed that insect axons provide a critical spatially restricted FGF-like signal that induces glia to enwrap them. (+info)
Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P(1) and P(1)' residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor.
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Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74-78). All these peptides are composed of 35-36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin (K(i) = 6.2 x 10(-12) M), and SGTI is a rather weak inhibitor of trypsin (K(i) = 2.1 x 10(-7) M). The replacement of P(1) then P(1)' residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor (K(i) = 5.0 x 10(-12) M) that retained some inhibitory affinity towards chymotrypsin (K(i) = 3.5 x 10(-8) M). The documented role of both P(1) and P(1)' highlights the importance of S(1)'P(1)' interactions in enzyme-inhibitor complexes. (+info)