Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Complexation of fibronectin with tissue transglutaminase

Previous work [Lorand, L., Dailey, J. E., & Turner, P. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1057-1059] showed that fibronectin might serve as a specific carrier for transglutaminases accidentally discharged from erythrocytes or other cells into plasma. In the present study we examined the association of these proteins in purified systems. Complexation was readily demonstrable by nondenaturing electrophoresis, using dansylcadaverine-dependent activity staining as well as immunoblotting procedures, and also by HPLC gel filtration. The results indicate a stoichiometry of 2:1 for the binding of the human erythrocyte transglutaminase (80K) to human plasma fibronectin (440K). The attachment is noncovalent in nature and does not involve cross-linking of the proteins either to themselves or to each other. Binding occurs in the absence of Ca2+, suggesting that a domain on the transglutaminase molecule other than the catalytic site is needed for complexation with fibronectin. Limited proteolysis with chymotrypsin for delineating the relevant region in fibronectin yielded two gelatin- (collagen) binding fragments (56K and 46K), each displaying affinity for transglutaminase. Moreover, these fragments--like intact fibronectin--bound erythrocyte transglutaminase and gelatin simultaneously in ternary complexes.     

Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.     

Light-temperature interactions on the growth of Antarctic diatoms

Summary The combined effect of various temperatures and light intensities on the growth of seven species of antarctic diatoms in culture has been studied. With the exception of Chaetoceros deflandrei whose thermal tolerance is fairly good, these obligatory psychrophils cannot survive in temperatures above 6° to 9° C. Their mean growth rate is relatively low, between 0.24 div d^–1 for Corethron criophilum and 0.63 div d^–1 for C. deflandrei. Regardless of light intensity, growth rate increased with the temperature to reach a maximum between 3° and 5° C. The highest rates were obtained between 115 and 220 mol m^–2 s^–1 with 0.38 div d^–1 for C. criophilum, 0.56 div d^–1 for Synedra sp. and between 0.71 and 0.88 div d^–1 for the other 5 species. A reduction in light intensity from 220 to 46 mol m^–2 s^–1 slowed growth by nearly 50%. These results suggest that the combined effect of temperature and light is one of the factors involved in the limitation of antarctic phytoplankton growth. The low temperatures of the environment do not permit rapid growth, which, even under optimal light conditions remains low. In addition, in the euphotic layer, the overall light energy available for algae is considerably reduced due to turbulence, a factor which exacerbates the reduced growth rate.     

Characterization of the muscarinic cholinergic receptor in the opossum (Didelphis virginiana, Kerr) submandibular gland: differences in receptor density and subtype compared with higher mammalian species

1. Kinetic, saturation and inhibition radioligand binding experiments with [3H]-N-methylscopolamine and [3H]quinuclidinyl benzilate were used to characterize the muscarinic cholinergic receptor in opossum (Didelphis virginiana, Kerr) submandibular salivary gland membranes. 2. The receptor density in opossum submandibular gland was found to be more than 3-fold higher than in rat, and 22-fold higher than in human, submandibular glands. 3. Inhibitor equilibrium dissociation constants for the antagonists pirenzepine, dicyclomine, atropine, N-methylscopolamine and AF-DX 116 revealed that the muscarinic receptor present in opossum submandibular gland appears to be the M1 subtype rather than the M3 subtype found in human and rat.     

Mutational analysis of the tobacco mosaic virus 5''-leader for altered ability to enhance translation.

Mutational analysis of the 5'-untranslated leader sequence (omega) of tobacco mosaic virus (TMV) was carried out to determine those sequences necessary for the translational enhancement associated with omega. Five deletion mutants, a single base substitution, and a 25 base replacement mutant were tested for alterations in omega's ability to enhance expression of beta-glucuronidase (GUS) mRNA in tobacco mesophyll protoplasts and Escherichia coli or chloramphenicol acetyltransferase (CAT) mRNA in Xenopus laevis oocytes. Alteration of an eight base subsequence required for the binding of a second ribosome resulted in the loss of translational enhancement in X. laevis oocytes but not in protoplasts. Substantial increases in enhancement were observed for several of the mutants in E. coli.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Taxonomy ofCarminatia (Asteraceae,Eupatorieae)

A taxonomic study of the largely Mexican genusCarminatia is rendered. It is comprised of three closely related species:C. tenuiflora, C. recondita andC. anomala spec. nova. Illustrations, dot maps, keys to species and complete synonymy are presented.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.