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PubMed1995

Antagonizing effect of morphine on the mobility and phagocytic activity of invertebrate immunocytes.

In the present study we have demonstrated that lipopolysaccharide (LPS) acts as an activator in the immunocytes of molluscs Planorbarius corneus, Mytilus edulis, and the insect Leucophaea maderae. This stimulatory effect, demonstrated by cellular conformational changes, is concentration- and time-dependent, and is antagonized by morphine. The inhibitory effect of morphine can be counteracted by naloxone. Morphine inhibitory action on immunocyte activity is also demonstrated by a decrease in the phagocytic activity. These data suggest that the downregulation of morphine is not limited to vertebrates but is also present in invertebrates.

Animals↗

PubMed1996

The use of invertebrate peptide toxins to establish Ca2+ channel identity of CA3-CA1 neurotransmission in rat hippocampal slices.

The relative contribution(s) of different Ca2+ channel subtypes to synaptic transmission between Schaffer collaterals of hippocampal CA3 pyramidal cells and CA1 pyramidal cell dendrites has been assessed using the synthetic invertebrate peptide toxins omega-conotoxin GVIA to block N-type Ca2+ channels, omega-agatoxin-IVA to block P-type Ca2+ channels and omega-conotoxin MVIIC to block N-, P- and Q-type Ca2+ channels. Omega-Agatoxin-IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC all produced dose-dependent inhibitions of the excitatory post-synaptic field potential (fEPSP) recorded from the CA1 region of transverse hippocampal slices. Application of 300 nM omega-conotoxin GVIA generally produced no further inhibition to that observed with 100 nM, resulting in a maximal 50% inhibition of the fEPSP. By contrast, 30 nM omega-agatoxin-IVA reduced the fEPSP slope by only 4.6 +/- 11.1% (mean +/- S.D., n = 3), suggesting the lack of involvement of classical P-type Ca2+ channels, whereas 300 nM omega-agatoxin-IVA reduced the fEPSP slope by 85.7 +/- 15.3% (n = 3) at the end of 44 min application. Similar applications of 100 and 300 nM sigma-conotoxin MVIIC reduced the fEPSP slope by 30.9 +/- 6.6% and 79.7 +/- 5.7% respectively. Application of 30 nM omega-agatoxin-IVA together with omega-conotoxin GVIA (300 nM) produced no greater inhibition of the fEPSP than that observed with omega-conotoxin GVIA alone, suggesting that the omega-agatoxin-IVA-sensitive and omega-conotoxin MVIIC-sensitive component presents a pharmacology similar to the reported Q-type Ca2+ channel. The inhibition produced by omega-conotoxin GVIA and omega-conotoxin MVIIC showed no recovery with prolonged washing (1-2 h) whereas that produced by omega-agatoxin-IVA was slowly reversible. The observation that omega-agatoxin-IVA, which does not effect N-type Ca2+ channels (Mintz et al. (1992a) Neuron 9, 85), is capable of completely suppressing the fEPSP suggests that, whilst N-type Ca2+ channels may contribute to normal synaptic transmission at Schaffer collateral-CA1 synapses, they are not capable of supporting transmission when Q-type channels are blocked.

Action Potentials↗

PubMed1992

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RGD-dependent.

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.

Amino Acid Sequence↗

PubMed1990

Invertebrate rhodopsin cleavage by an endogenous calcium activated protease.

Calcium-activated proteases have been purified from a number of vertebrate tissues, including the retina and lens. These proteases exhibit similar characteristics and are believed to be involved in the regulation of cytoskeletal elements. Here we report the partial purification and characterization of a calcium-activated protease from the squid photoreceptor cell which, when activated, specifically removes 10 kDa from the carboxyl-terminal of squid rhodopsin. No other detectable soluble proteins from the invertebrate photoreceptor are susceptible to cleavage and only one non-opsin, integral membrane protein shows evidence of cleavage. The enzyme requires 5 mM calcium for half maximal activation, and is not significantly activated by other divalent ions. The protease has a molecular weight of approximately 350 kDa, as determined by gel filtration, and when partially purified by casein affinity chromatography, it runs as three main bands of 76, 63 and 36 kDa on SDS-PAGE. The crude protease loses as much as 80% of its activity in 24 hr, whereas the partially purified protease is stable up to 4 weeks at 4 degrees C.

Amino Acid Sequence↗

PubMed1986

Phylogenetic comparison of lens crystallins from the vertebrate and invertebrate--convergent or divergent evolution?

A systematic biochemical comparison has been made of the crystallins isolated from the lenses of five different species belonging to the five major classes of vertebrates. Gel-permeation chromatography of the lens homogenates on Fractogel TSK HW-55(S) revealed well-defined elution patterns with a characteristic distribution of different classes of crystallins from each species. SDS gel electrophoresis and statistical comparison of the amino acid contents indicated that all crystallin groups from different classes share some common subunits and similarity in their amino acid compositions. The results coupled with the relatedness shown in the amino acid compositions of fish gamma-crystallin with those of mammalian gamma-crystallin and the squid crystallin from the invertebrate pointed to the possibility of the existence of a common ancestral protein for all crystallins. This is in favor of the divergent rather than convergent evolution of lens crystallins as commonly assumed in the literature.

Amino Acid Sequence↗

PubMed1992

Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor.

We have previously reported [(1991) EMBO J. 10, 3239-3245] the sequence of an invertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo-oligomeric GABA-gated, bicuculline-sensitive, chloride-ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5-4864 (4'-chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly active the homo-oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.

Animals↗

PubMed1984

Influence of ionic environment of the stress relaxation behavior of an invertebrate connective tissue.

The rheological properties of an invertebrate connective tissue were measured in three different ionic environments. Short-term stress relaxation tests were conducted on sections of holothurian (Echinodermata) body wall immersed in isotonic monovalent and divalent salt solutions and deionized water. Using a reduced modulus format, the viscoelastic behavior over the experimental time scale was described by a two term Maxwell equation with empirically determined spring constants and relaxation times. In addition, equilibrium relaxation moduli (Ge) were estimated from the empirical relationship of Chasset and Thirion (1965, in Physics of Non Crystalline Solids, ed. Prins, North Holland). The experiments indicated that both relaxation times and equilibrium moduli decreased in the presence of monovalent and divalent inorganic ions whereby the effect of the Na+ was greater than that of the Ca++. The present findings are compared with those reported for vertebrate connective tissue.

Animals↗

PubMed1989

Overall study of the in vitro plasma clotting system in an invertebrate, Liocarcinus puber (Crustacea Decapoda): considerations on the structure of the Crustacea plasma fibrinogen in relation to evolution.

An overall study of the in vitro plasma coagulation system in the crab Liocarcinus puber has been carried out using various analytical methods, namely thromboelastography, spectrophotometrical examination, and a new one based on changes of the mechanical impedance of the developing clot. From the results reported here the clotting pattern in this species appears surprisingly complex for an invertebrate and unexpectedly closer to that of the vertebrates. Indirect evidences suggest that the fibrinogen polypeptide chains in this species and very likely in the other crustacean, are very different from those of the vertebrates. This would imply that crustacean and vertebrate fibrinogen would have diverged from one another in a far remote past, far beyond the individualization of the vertebrate alpha chain, that is, over 1.5 million years ago.

Animals↗

PubMed1987

The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments.

We establish that the position-specific antigen 2 (PS2), a Drosophila cell surface glycoprotein complex, is an invertebrate member of the vertebrate fibronectin receptor (integrin) family. New monoclonal antibodies show that in Drosophila embryos and larvae PS2 alpha subunits have a size of ca. 140 kd. Analysis of cDNA and genomic clones revealed that the canonical PS2 alpha subunit contains 1394 amino acids and has extensive homology to the heavy and light chains of integrin alpha subunits. The distribution of the PS2 antigen is regulated at the level of PS2 alpha subunit mRNA. In early Drosophila development the protein is restricted to mesoderm and appears to be involved in muscle attachment. We suggest that PS2, like vertebrate fibronectin receptors, mediates changes in cell shape and cell-extracellular matrix adhesion by binding to a basement membrane protein.

Amino Acid Sequence↗

PubMed1990

Immunomodulatory stimulation of an invertebrate circulatory lectin by its haptenic molecules of pathogenic origin.

2-keto-3-deoxy octonate and beta-glycerophosphate two important bacterial cell wall constituent molecules, haptenic in nature to an invertebrate (crab) circulatory lectin, carcinoscorpin, gave lectin induction after immunization of the crab, Carcinoscorpius rotunda-cauda. With the exception of erythrocyte, no other sialoglycoconjugate-containing substances (mucin, fetuin) sialodisaccharide O-[N-acetylneuraminyl-(2----6)2-acetamido-2-deoxy galactitol] and free sialic acid were effective in lectin induction. This induction of circulatory lectin failed to appear when, concanavalin A, epinephrine, cytochalasin B, methyl-alpha-D-mannoside and bovine serum albumin were used for immunization. But mechanical injury to crabs were extremely effective in such lectin induction by live crabs. LPS is lethal if used for immunization. The immunoregulatory induction of the circulatory lectin by pathogen-originated cell wall constituent molecules and mechanical injury may be considered as a humoral immune response.

Animals↗

PubMedDate not supplied

An invertebrate (Molluscan) plasma protein that binds to vertebrate immunoglobulins and its potential for yielding false-positives in antibody-based detection systems.

Commercial immunoassay kits that incorporate monoclonal antibodies are being used increasingly to screen tissues and plasma from invertebrates for the presence of antigens that are taken to be homologues of the vertebrate immunogen. We report the presence of a protein (BgL115r) in the plasma of Biomphalaria glabrata that binds to immunoglobulins from a variety of vertebrates. BgL115r (ca. 230 kD non-reduced, ca. 115 kD reduced on SDS-PAGE) binds to the F(ab')2-region of murine IgG. Binding could be inhibited with high concentrations of N-acetyl-D-glucosamine (GlcNAc). BgL115r was found to yield false positive signals in an ELISA based assay.

Animals↗

PubMed1988

Phototaxis in aquatic invertebrates: possible use in ecotoxicity tests.

A series of experiments has been carried out aimed at (i) a deeper study of the phototaxis of some aquatic invertebrates and (ii) its possible use in ecotoxicity tests. For the first point the phototaxis of the nauplii of Artemia salina was examined: through tests carried out under different conditions, it was observed that a marked complex response to light existed, which led to a subdivision between individuals with different phototactic response, mainly depending on the density of the nauplii, on light intensity, and on spectral composition. The second point has been analyzed with A. salina, Daphnia magna, and Aëdes aegypti. For the first species it must be stressed that in ecotoxicity tests carried out with nauplii of A. salina one should bear in mind the complexity of their phototactic response. With D. magna and A. aegypti a simple pvc apparatus connected with optic fibers and set up on a microscope was used. The result is marked and easily observable; therefore, it will be possible in the future to perform easily tests with toxic substances which are important in ecotoxicology.

Aedes↗

PubMed1990

Field deployment of a scope for growth assay involving Gammarus pulex, a freshwater benthic invertebrate.

Scope for growth (SfG) is a measure of the energy balance of an animal (i.e., the difference between energy intake and metabolic output). The SfG of marine invertebrates, particularly the mussel Mytilus edulis, has been successfully used as the basis of a field bioassay to detect a range of stresses both natural (temperature, food, salinity) and anthropogenic (hydrocarbons, sewage sludge). SfG of the freshwater amphipod Gammarus pulex was found to be a sensitive indicator of stress under laboratory conditions and here we describe the field deployment of this technique and present data from three field trials. In every case, SfG was reduced at the downstream polluted site compared with that at an upstream reference site. This reduction in SfG was the result of a decrease in energy intake (absorption) rather than an increase in energy expenditure (respiration).

Animals↗

PubMed1989

Sealing cultured invertebrate neurons to embedded dish electrodes facilitates long-term stimulation and recording.

Recently it has become possible to form small networks of synaptically connected identified invertebrate neurons in culture. Using conventional saline-filled glass electrodes, it is difficult to simultaneously stimulate and record from more than 2 or 3 cultured neurons and to perform experiments lasting longer than several hours. We demonstrate that it is possible to overcome these limitations by using planar arrays of electrodes embedded in the bottom of a culture dish. The arrays employ conductive leads and insulation that are transparent, making the dishes compatible with voltage-sensitive dyes and inverted microscopy. Identified neurons from leech Hirudo medicinalis, slug Aplysia californica, and snail Helisoma trivolvis, have been grown on these arrays. Due to their large size (soma diameter 40-200 microns) these neurons form seals over the dish electrodes. Individual electrodes can then be used to stimulate and to record action potentials in the associated neuron. With sealing, action potentials have been recorded simultaneously from many neurons for up to two weeks, with signal-to-noise ratios as large as 500:1. We developed and tested a simple model that describes the voltage waveforms measured with array electrodes. Potentials measured from electrodes under cell bodies were primarily derivatives of the intracellular potential, while those measured from electrodes under axon stumps were primarily proportional to local inward Na+ currents. While it is relatively easy to record action potentials, it is difficult to record postsynaptic potentials because of their small size and slow rate of rise.

Action Potentials↗

PubMed1991

A method for the determination of projection areas of GABA immunoreactive neurons in the invertebrate nervous system.

Axonal transport of metallic salts (nickel or cobalt chloride) has been widely used for the anatomical mapping of neural pathways. We show here that when nickel is introduced into GABAergic neurons it completely eliminates GABA immunolabelling. We have used this property to determine the axonal projections of GABAergic neurons in the stomatogastric system of Crustacea. For example, following nickel backfills from either cut axons or from terminals, GABA immunostaining labels only those GABA-immunoreactive neurons which had not been retrogradely labelled with nickel and hence did not project in the cut nerve or to the neuropile uptake site. By comparing such immunolabelled preparations with those not pretreated with nickel the projection patterns of all the GABA immunoreactive neurons in a given system can be revealed. This effect of nickel appears to be selective for GABA immunostaining, insofar as it does not interfere with the immunodetection of either the peptide proctolin or a FMRFamide-like peptide. This method may prove to be a useful tool for analyzing GABAergic neuronal pathways in the nervous systems of invertebrates.

Animals↗

PubMed1995

Effect of prolonged exposure to morphine on responsiveness of human and invertebrate immunocytes to stimulatory molecules.

This study deals with a novel role of morphine in the modulation of cellular responsiveness to immunostimulatory substances that, at first glance, appears to be in contrast to the well documented immunoinhibitory short-term effects of opiate alkaloids on cells simultaneously exposed to stimulatory molecules. Vertebrate and invertebrate immunocytes pre-exposed to morphine (10(-6) M) in vitro for at least 24 h prior to the administration of lipopolysaccharide (LPS; 1.0 micrograms/ml) or other immunoactivating substances have revealed a distinct enhancement of their responsiveness to these signals, e.g. monocytes exposed to LPS alone resulted in 21% activation, whereas the morphine pretreated level was at 40% (P < 0.01). Prolonged pretreatment with morphine of naive human monocytes had the same effect on their sensitivity to plasma from patients having undergone cardiopulmonary bypass (CPB) operations followed by a diffuse inflammatory response. These results suggest that endogenous opiates may participate, in more than one way, in re-establishing an organism's readiness to meet a new demand on its immune system. Additional support for the concept of a role of endogenous opiates in immunomodulation was obtained by the results of in vivo tests with experimentally induced stress in Mytilus. Following their stress-induced stimulation, these animals' immunocytes could be shown to become exposed for some time to a measurable rise in endogenous morphine-like material (9 pmol/ml increasing to 59). These immunocytes, like those preincubated with exogenous morphine, displayed a heightened sensitivity to stimulation by LPS (control 21.3 +/- 3.1% activation compared to 47.2 +/- 5.1) when the morphine levels dropped. The mechanism of this enhancement of responsiveness to immunostimulation following the prolonged exposure of immunocytes to morphine, and its relationship with the known short-term immunoinhibitory opiate effects on the immune system, remains to be ascertained.

Animals↗

PubMed1993

Identification of homologues to beta-catenin/plakoglobin/armadillo in two invertebrates, Urechis caupo and Tripneustes gratilla.

beta-Catenin and plakoglobin are intracellular proteins that participate in cell-cell adhesion, probably through interaction with the cadherin family of transmembrane adhesion proteins. They are also homologous to the segment polarity gene, armadillo, from Drosophila. I have cloned and sequenced armadillo/beta-catenin/plakoglobin homologues from two other invertebrates, Urechis caupo and Tripneustes gratilla, and shown that the mRNA is present in oocytes, eggs and early embryos. In Urechis, the mRNA is particularly abundant in oocytes, but is not translated until after fertilization. These results provide further indications that cell adhesion proteins play a key role during embryogenesis.

Amino Acid Sequence↗

PubMed1993

Levels of ethanolamine intermediates in the human and rat visual system structures: comparison with neural tissues of a lower vertebrate (Mustelus canis) and an invertebrate (Loligo pealei).

Levels of ethanolamine intermediates in the retina and optic nerves of autopsied human donors and in the rat visual system (retina, optic nerve, lateral geniculate body, superior colliculus) were measured. Amounts were also obtained from the retina, optic nerve, and optic tectum of a primitive elasmobranch, the smooth dogfish Mustelus canis, and from the related nervous structures (retina, optic lobe, fin nerve, and stellate ganglia) of a marine invertebrate, the squid Loligo pealei. In all regions of the human and rat nervous system, the pool size of CDP-ethanolamine (values ranging between 10-31 nmol/g wet wt) was much smaller than that of free ethanolamine (values ranging between 197-395 nmol/g wet wt), whereas glycerophosphorylethanolamine was present in relatively high content (values ranging between 125-280 nmol/g wet wt). In nervous system regions of the dogfish and squid, the distribution of values followed the same general trend as observed for humans and rats, even if all regions had less ethanolamine intermediates compared to the mammalian counterpart. In dogfish and squid retina, glycerophosphorylethanolamine showed the highest pool size among the ethanolamine derivatives analyzed (16 and 44 nmol/g wet wt, respectively). The present study confirms the basic similarity of ethanolamine intermediate pool size patterns in the nervous system structures (with the exception of the retina) of animal species which have widely different phylogenetic positions. The data support the proposal that the levels reached by ethanolamine and its derivatives in the nervous tissue is the result of an ancient evolutionary development of metabolic pathways for the maintenance of phosphatidylethanolamine membraneous content.

Adolescent↗