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Structure-function relationships of pheromones of the ciliate Euplotes raikovi with mammalian growth factors: cross-reactivity between Er-1 and interleukin-2 systems.

Diffusible protein signals of the ciliate Euplotes raikovi, denoted as pheromones, have functionally been linked with prototypic growth factors of animal cells by the demonstration that they not only induce a temporary cell union in mating pairs, by acting in a paracrine-like fashion, but can also bind to cells in autocrine fashion and promote their vegetative (mitotic) proliferation. It is now shown that pheromone Er-1 is capable of binding to the alpha and beta chains of the multimeric IL-2 receptor on mammalian cells and that IL-2 can, in turn, bind to the putative cell receptor of this pheromone. Similarities in the IL-2 and Er-1 structures support these findings and raise controversial implications with regard to their evolutionary significance.

Amino Acid Sequence↗

Chairman's summary: mechanisms of oxygen homeostasis, circa 1999.

Oxygen sensing is a fundamental physiologic requirement of all living organisms and all cells within the human body. This paper presents a brief summary of recent investigations into the molecular mechanisms underlying oxygen sensing and adaptive responses to hypoxia, with particular reference to other papers in this volume.

Animals↗

Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway.

This study applies biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent superoxide anion (O2-) formation and localisation of hydroxyl radicals (*OH) by 3-dimensional (3D) confocal laser scanning microscopy to reveal in human cells putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia. A cell membrane localised non-mitochondrial cytochrome b558 seems to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563 probably probing additionally metabolic changes. *OH the putative second messenger of the oxygen sensing pathway generated by a Fenton reaction could be visualized in the perinuclear space of the three human cell lines used. Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia induced gene expression, interact on various sides of the oxygen sensing pathway confirming the importance of b-type cytochromes and the Fenton reaction.

Chemoreceptor Cells↗

Are odorant-binding proteins involved in odorant discrimination?

Pheromone-sensitive sensilla trichodea of nine moth species belonging to six families and three superfamilies of Lepidoptera were immunolabelled with an antiserum against the pheromone-binding protein of Antheraea polyphemus. Strong immunolabelling of the sensillum lymph was observed in all long sensilla trichodea of A. polyphemus, A. pernyi (Saturniidae), Bombyx mori (Bombycidae) and Manduca sexta (Sphingidae). Very weak labelling was found with all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae) and Lymantria dispar (Lymantriidae). In three noctuid species, some long sensilla trichodea were labelled strongly, some only weakly and some were not labelled at all. The fraction of long sensilla trichodea that were strongly labelled was large in Helicoverpa armigera, but small in Spodoptera littoralis and Autographa gamma. The observed cross-reactivity was not correlated with taxonomic relatedness of the species but rather with chemical relatedness of the pheromones used by these species, as a high labelling density was consistently observed in sensilla tuned to pheromones with an alcyl chain of 16 carbon atoms. The highly divergent specificity of pheromone-receptor cells in Noctuidae appears to be mirrored by a similar diversity of the pheromone-binding proteins in the sensilla trichodea. These data support the notion that pheromone-binding proteins participate in odorant discrimination.

Animals↗

Effects of adaptation in maintaining high sensitivity over a wide range of backgrounds for Escherichia coli chemotaxis.

An allosteric model is developed to study the cooperative kinase response of wild-type (wt) Escherichia coli cells to the chemoattractant MeAsp in different ambient MeAsp concentrations. The model, together with wt dose response data, reveals the underlying mechanism for E. coli's ability to maintain high sensitivity over a wide range of backgrounds. We find: 1), Adaptation tunes the system to the steepest part of the dose response curve, where the sensitivity to a given type of stimulus is amplified by the number of corresponding receptors in the (mixed) functional receptor complex. A lower bound on the number of Tar receptor dimers (Na) in the complex Na>approximately 6 is obtained from the measured sensitivity. 2), Accurate adaptation synchronizes the kinase activities from different (uncoupled) receptor complexes in a single cell and is crucial in maintaining the high Hill coefficient in the (population averaged) kinase response curve. 3), The wide dynamic range of the high sensitivity can be explained in our model by either having a very small ratio between ligand dissociation constants of the inactive and the active receptors C=0.006, Na=6, and a (methylation level independent) dissociation constant for the inactive Tar receptor K=18.2 microM or by having K and/or Na increase with receptor methylation level together with a larger value of C>0.01. Specific experiments are suggested to distinguish these two scenarios. 4), The receptor occupancy in a wt cell should also adapt and exhibit a slow (approximately logarithmic) dependence on the ligand concentration in the adapted state; this general prediction can be tested experimentally to verify/falsify our model.

Adaptation, Physiological↗

Cortical unit responses to chemical stimulation of the oral cavity in the rat.

The gustatory sensitivity of individual cortical neurons in the rat was examined using a wide range of chemical stimuli. Several subsets of cortical taste cells were identified based on similarities in their response profiles. Although adjacent cortical neurons can differ in their responsiveness to individual tastants, some overlap in taste sensitivities is usually observed.

Afferent Pathways↗

Molecular cloning of ictacalcin: a novel calcium-binding protein from the channel catfish, Ictalurus punctatus.

Calcium is essential for a variety of functions in animals, including signal transduction, transmission of nerve impulses, and bone and scale growth. In freshwater adapted teleosts, blood calcium levels are maintained constant (2-4 mM) even at low external calcium concentration (< 0.01 mM). Epithelial cells in skin and gill have been implicated in calcium homeostasis. We have cloned a cDNA from Ictalurus punctatus, the channel catfish, that codes for ictacalcin, a novel member of the S100 family of calcium-binding protein. In-situ hybridization demonstrates ictacalcin mRNA is abundant in epithelial cells of olfactory rosette, barbel, skin and gill but not brain or muscle. The presence of ictacalcin protein in these tissues was confirmed by immuno-blot analysis. Tissue extracts and recombinant ictacalcin bind calcium with attendant changes in electrophoretic mobility indicative of changes in protein conformation. The calcium-binding activity and abundant localization of ictacalcin in epithelial cells of several tissues indicates that this protein plays an important role in catfish calcium homeostasis.

Amino Acid Sequence↗

Stimulus-specific mobilization of dopamine and norepinephrine stores in cat carotid body.

The catecholamines (CAs), dopamine (DA) and norepinephrine (NE), are synthesized and stored in carotid body chemosensory type I cells. Previous studies in our laboratory demonstrated that low concentrations of nicotine preferentially evoke the release of NE from rabbit type I cells, whereas hypoxia mobilizes DA and NE in proportion to their stores in the tissue. The primary objective of the present study was to examine whether hypoxia, nicotine and elevated concentrations (30 mM) of K+ evoke the preferential release of DA vs. NE from cat carotid bodies superfused in vitro. In this species, where tissue stores of DA and NE are nearly equal, hypoxia evoked the preferential release of DA from normal carotid bodies. This pattern of release evoked by low O2 was also present following chronic removal of the superior cervical ganglion, which eliminated NE contained in the sympathetic innervation to the carotid body. In contrast, nicotine and high-K+ preferentially mobilized NE in these sympathectomized animals. Sympathectomy also reduced the percent of DA (but not NE) content released from type I cells in response to any of the three stimuli. Our findings suggest that chemosensory type I cells possess stimulus-specific mechanisms for CA mobilization and that the sympathetic innervation modulates the metabolism and release of CAs in the cat carotid body.

Animals↗

Carbon monoxide: a role in carotid body chemoreception.

Carbon monoxide (CO), produced endogenously by heme oxygenase, has been implicated as a neuronal messenger. Carotid bodies are sensory organs that regulate ventilation by responding to alterations of blood oxygen, CO2, and pH. Changes in blood gases are sensed by glomus cells in the carotid body that synapse on afferent terminals of the carotid sinus nerve that projects to respiratory-related neurons in the brainstem. Using immunocytochemistry, we demonstrate that heme oxygenase 2 is localized to glomus cells in the cat and rat carotid bodies. Physiological studies show that zinc protoporphyrin IX, a potent heme oxygenase inhibitor, markedly increases carotid body sensory activity, while copper protoporphyrin IX, which does not inhibit the enzyme, is inactive. Exogenous CO reverses the stimulatory effects of zinc protoporphyrin IX. These results suggest that glomus cells are capable of synthesizing CO and endogenous CO appears to be a physiologic regulator of carotid body sensory activity.

Analysis of Variance↗

A scanning and transmission electron microscope study of the premetamorphic papillae: possible chemoreceptive organs in the oral cavity of an anuran tadpole (Rana japonica).

Premetamorphic papillae of an anuran tadpole (Rana japonica) were studied by scanning and transmission electron microscopy. Premetamorphic papillae of several shapes are seen in and around the oral cavities of tadpoles during late larval and early metamorphic stages. These papillae are composed of three parts: the apical cellular part, the underlying connective tissue core and the epithelial covering. In the apical cellular part, two populations of cells are observed: apical and basal cells. The apical cell has a slender cytoplasmic process whose apical surface is exposed in the oral cavity. The basal cell is located at the basal portion of the apical cellular part of the papilla without researching the oral cavity. A long solitary microvillus and a bunch of short microvilli are seen on the apical surface of each apical cell. The apical cells contain dense-cored vesicles of 100 nm diameter and make synaptic contacts at their basal membranes with terminals of nerve fibers. Possible chemoreceptive function of the premetamorphic papilla is discussed.

Animals↗

Neuronal uptake of pesticides disrupts chemosensory cells of nematodes.

Low doses of the acetylcholinesterase-inhibiting carbamate nematicides disrupt chemoreception in plant-parasitic nematodes. Fluorescein isothiocyanate (FITC)/dextran conjugates up to 12 kDa are taken up from the external medium by certain chemosensory neurons in Caenorhabditis elegans. Similar chemoreceptive neurons of the non-feeding infective stage of Heterodera glycines (soybean cyst nematode) fill with FITC and the nuclei of their cell bodies selectively stain with bisbenzimide. The widely used nematicide aldicarb disrupts the chemoreceptive response of H. glycines with 50% inhibition at very low concentrations (ca 1 pM), some 10(-6)-fold lower than required to affect locomotion. Similarly, the anthelmintic levamisole had this effect at 1 nM. Peptides selected as mimetics of aldicarb and levamisole also disrupt chemoreception in H. glycines and Globodera pallida at 10(-3)-fold or lower concentration than required to inhibit locomotion. We propose an uptake pathway for aldicarb, levamisole, peptide mimetics and other soluble molecules by retrograde transport along dendrites of chemoreceptive neurons to the cell bodies and synapses where they act. This may prove to be a general mechanism for the low-dose effects of some nematicides and anthelmintics.

Aldicarb↗

[Role of cyclic nucleotides in carotid chemoreception].

The activation of the metabolic systems of rat carotid body under the influence of alkaloids and acids was studied by spectrofluorimetric technique. It has been demonstrated the growth of the cAMR levels being suggested to the role of messenger between chemical stimuli and the flows of glomus cells metabolism. Chemical substances of other types (such as acetylcholine, NaCl saccharose) don't provide any changes of cell respiration and concentration of cAMP in carotid cells. The data obtained provide the evidence of heterogeneity of glomus cella reception mechanisms of the qualitatively different chemical substances. It supports the hypothesis on the heterogeneity of chemoreception for mammals.

Acetylcholine↗

[Ultrastructure of the surface of the osphradium of the anterior gilled mollusk Murex saxatilis L].

The Murex saxatilis L. (Mollusca, Prosobranchia) osphradium is of the most complex "ctenidial" type and is situated immediately behind the siphon that brings water to the mantel cavity. By means of scanning and transmissive electron microscopical investigation, three zones have been revealed on the surface of the osphradium petals: secretory, intermediate and receptory. The secretory zone occupies the lateral part of the petal and is formed with 1-2 layers of cells. The intermediate zone, as a narrow stria, is situated between the secretory and the receptory zones. Here, together with the secretory cells, the ciliary cells are present. The receptory zone occupies nearly the whole surface of the petal. It is much thicker and is formed of pseudostratified epithelium where 5-7 raws of nuclei can be counted. Besides the cell types mentioned, there are defined bipolar receptory cells in it. The apical surface of the receptory cells has up to 200 cilia, and fine peripheral processes--1-2 cilia. Presence of the complex receptory zone makes it possible to suggest certain differentiation of the stimuli already at the peripheral level of the sensory system.

Animals↗

Chemo- and baroreceptor innervation of the aortic trunk of the toad Bufo vulgaris.

In the toad Bufo vulgaris, afferent impulses were recorded from the fine branch of the vagus which terminates in the aortic trunk. Spontaneous single unit discharge was random. When the aorta was artificially perfused, units fired in response to flow change, hypoxia and hypoxia-hypercapnia of the perfusion fluid, and administration of NaCN or Ach. A few baroreceptor fibers fired synchronously with the blood pressure fluctuation. In anesthetized toads with other reflexogenic areas denervated, the intravenous administration of NaCN caused an increase in breathing and a slight elevation of blood pressure. When the nerve concerned was cut, these responses were abolished. Electrical stimulation of the nerve's central cut end brought about the same type of response as that to NaCN. Special cells closely resembling the glomus cells in the carotid labyrinth were found in the aortic wall. In amphibia, there is an aortic nerve corresponding to that of mammals, and the aorta is one of receptive areas predominantly contributing to respiratory control.

Animals↗

Reduced glutathione, dithiothreitol and cytochrome P-450 inhibitors do not influence hypoxic chemosensory responses in the rat carotid body.

Glomus cells and carotid sinus afferents are anatomically connected, and the chemical events in the glomus cells are expected to be conveyed reflexly as afferent signals. Accordingly, K(+) channel inhibition of the glomus cell membrane is expected to be followed by excitation of the afferents. In order to test the redox inhibition of K(+) channels of glomus cells by reduced glutathione (GSH), dithiothreitol (DTT) and by cytochrome P-450 inhibitors (clotrimazole and miconazole), we measured the carotid sinus nerve (CSN) discharge using an in vitro perfused adult rat carotid body (CB) in the presence and absence of these chemicals which are expected to excite the afferents. Our findings were that these agents did not stimulate the CSN activities during normoxia and kept the hypoxic responses intact. These results led us to conclude that the redox modulation of glomus cells was not conveyed to the afferents, and this functional disconnection did not support the redox hypothesis of O(2) chemoreception in the whole carotid body.

Animals↗

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Acyltransferases↗

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats.

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.

Amino Acid Sequence↗

Role of gap junctions in CO(2) chemoreception and respiratory control.

Gap junctions are composed of connexins, which are organized into intercellular channels that form transmembrane pathways between neurons (cell-cell coupling), and in some cases, neurons and glia, for exchange of ions and small molecules (metabolic coupling) and ionic current (electrical coupling). Cell-cell coupling via gap junctions has been identified in brain stem neurons that function in CO(2)/H(+) chemoreception and respiratory rhythmogenesis; however, the exact roles of gap junctions in respiratory control are undetermined. Here we review the methods commonly used to study gap junctions in the mammalian brain stem under in vitro and in vivo conditions and briefly summarize the anatomical, pharmacological, and electrophysiological evidence to date supporting roles for cell-cell coupling in respiratory rhythmogenesis and central chemoreception. Specific research questions related to the role of gap junctions in respiratory control are suggested for future research.

Animals↗